OCS Study 

•

MMS 2002-050 

Effects of Produced Water on 

Complex Behavior Traits of 

Invertebrate Larvae 

Final Technical Summary 

Final Study Report 

U.S. Department of the Interior 

Minerals Management Service

Pacific OCS Region 

Effects of Produced Water on 

Complex Behavior Traits of 

Invertebrate Larvae 

Final Technical Summary 

Final Study Report 

Authors 

Peter Raimondi 

Principal Investigator

and 

Anthony Boxshall 

Prepared under MMS Cooperative 

Agreement No. 14-35-0001-30758 

by 

Coastal Marine Institute 

Marine Science Institute 

University of California 

Santa Barbara, CA 93106 

U.S. Department of the Interior 

Minerals Management Service Camarillo

Pacific OCS Region January 2002
Disclaimer 

This report has been reviewed by the Pacific Outer Continental Shelf Region, Minerals 

Management Service, U.S. Department of the Interior and approved for publication. The 

opinions, findings, conclusions, or recommendations in this report are those of the authors, and 

do not necessarily reflect the views and policies of the Minerals Management Service. Mention 

of trade names or commercial products does not constitute an endorsement or recommendation 

for use. This report has not been edited for conformity with Minerals Management Service 

editorial standards. 

Availability of Report

Extra copies of the report may be obtained from: 

U.S. Dept. of the Interior 

Minerals Management Service 

Pacific OCS Region 

770 Paseo Camarillo 

Camarillo, CA 93010 

Phone: 805-389-7621 

A PDF file of this report is available at: 

http://www.coastalresearchcenter.ucsb.edu/CMI/ 

Suggested Citation 

The suggested citation for this report is: 

Raimondi, P. T. and Boxshall A. Effects of Produced Water on Complex Behavior Traits of 

Invertebrate Larvae. MMS OCS Study 2002-050. Coastal Research Center, Marine Science 

Institute, University of California, Santa Barbara, California. MMS Cooperative Agreement 

Number 14-35-0001-30758. 38 pages. 
Table of Contents 

FINAL TECHNICAL SUMMARY....................................................................... 1 

 FINAL STUDY REPORT ..................................................................................... 4 

Introduction....................................................................................................... 4 

Methods.............................................................................................................. 6 

 General Comments........................................................................................ 6 

 Produced Water............................................................................................. 6 

 Organisms...................................................................................................... 7 

 Specific Methods........................................................................................... 8 

Results ................................................................................................................ 14 

 General Comments........................................................................................ 14 

 Specific Results............................................................................................. 14 

Discussion........................................................................................................... 34 

References.......................................................................................................... 37 
List of Tables 

Table 1 A summary of the experimental conditions for W. subtorquata....................... 9 

Table 2 W. subtorquata swimming behaviors. Swimming 75 minutes after 

 being washed from PW ........................................................................... 15 

Table 3 W. subtorquata larval metamorphosis 23 hours after being washed from PW 15 

Table 4 Survival of W. subtorquata colonies to the final day in Experiment 3............. 15 

Table 5 W. subtorquata survivorship to outplanting in Experiment 3 ........................... 16 

Table 6 W. subtorquata survivorship to outplanting in Experiment 3.......................... 16 

Table 7A Proportion of W. subtorquata larvae attached after 75 minutes of exposure ... 17 

Table 7B Proportion of W. subtorquata larvae metamorphosed after 23 hours 

 of constant exposure................................................................................ 17 

Table 8 The percentage of W. subtorquata larvae showing each behavior .................. 18 

Table 9 W. subtorquata survival from Day 20 to Day 40.............................................. 20 

Table 10 Repeated Measures of W. subtorquata growth rates ........................................ 20 

Table 11 The size of W. subtorquata zooids in Experiment 1 at day 150 ....................... 21 

Table 12 Growth rates of W. subtorquata throughout Experiment 2............................... 24 

Table 13 The size of W. subtorquata colonies in Experiment 3 at outplanting............... 24 

Table 14 Growth rates of W. subtorquata throughout Experiment 3............................... 24 

Table 15 Average W. subtorquata zooid size at the final census date............................. 25 

Table 16 The size of S.unicornis colonies at outplanting to the field.............................. 28 

Table 17 The size of Bugula neritina colonies at outplanting to the field ...................... 30 

Table 18 The size of Bugula neritina colonies after 70 days in the field........................ 30 

Table 19 The size of Bugula neritina colonies after 70 days in the field........................ 31 

Table 20 The proportional settlement of abalone larvae in four PW treatments............. 32 

List of Figures 

Figure 1 Proportion of Watersipora subtorquata swimming during exposure to PW.... 18 

Figure 2 Adult growth in the field for W. subtorquata ................................................... 22 

Figure 3 The final size of W. subtorquata colonies at Day 150...................................... 23 

Figure 4 Competitive interactions of W. subtorquata with colonial ascidians ............... 26 

Figure 5 The proportion of S. unicornis larvae swimming and metamorphosed 

 at different times after exposure to PW .................................................. 27 

Figure 6 The size of Schizoporella unicornis colonies at outplanting............................ 28 

Figure 7 Proportion of Bugula neritina larvae swimming after exposure to PW............ 29 

Figure 8 The size of Bugula neritina colonies after 70 days in the field........................ 31 

Figure 9 The density of ovicells present on Bugula neritina colonies............................ 32 

Figure 10 The proportional settlement of abalone larvae in four PW treatments............. 33 
Effects of Produced Water on Complex Behavior Traits of Invertebrate Larvae 

1

FINAL TECHNICAL SUMMARY 

STUDY TITLE: Effects of Produced Water on Complex Behavior Traits of Invertebrate 

Larvae

REPORT TITLE: Effects of Produced Water on Complex Behavior Traits of Invertebrate 

Larvae

CONTRACT NUMBER: 14-35-0001-30758 

SPONSORING OCS REGION: Pacific

APPLICABLE PLANNING AREA: Southern California

FISCAL YEAR(S) OF PROJECT FUNDING: FY 96, FY 97, FY 98 

COMPLETION DATE OF THE REPORT: December 2001 

COST(S): FY 96 - $25,000; FY 97 - $33,910; FY 98 - 63,819, FY 99 – no cost 

CUMULATIVE PROJECT COST: $122,729 

PROJECT MANAGER: Peter Raimondi

AFFILIATION: University of California, Santa Cruz

ADDRESS: Department of Biology, University of California, Santa Cruz, CA 95460 

PRINCIPAL INVESTIGATOR: 

1

Peter Raimondi 

POST DOCTORAL RESEARCHER: 1,2Anthony Boxshall

ADDRESSES: 1

Department of Biology, University of California, Santa Cruz, CA 95460; 

2

Marine Sciences Unit, EPA Victoria, Ernest Jones Drive, Macleod VIC, Australia, 3085 

KEY WORDS: ecotoxicology, produced water, oil drilling, invertebrate larvae, bryozoan, 

Watersipora spp, Bugula neritina, Haliotis rufescens, Schizoporella unicornis, sub-lethal impact, 

pulsed exposure, carry-over effects. 

BACKGROUND: Produced Water (PW) is a by-product of oil drilling and in the Southern 

California region a common form of disposal of PW is by direct discharge into the ocean from a 

platform-based diffuser. Many compounds and elements in PW are known to have toxic effects 
Final Technical Summary – Raimondi and Boxshall 

2

on a number of organisms however these toxic effects are not always lethal, even in static 

conditions. 

Short, spiked exposure sees larvae exposed to a pulse of PW for a short period of time and is 

unlikely to produce lethal effects, however it may result in a series of important sub-lethal effects 

for organisms in various stages of their lifecycles. It is now generally believed that the discharge 

of produced water can cause severe, generally sub-lethal, effects to organisms over distances well 

beyond that predicted by plume dilution models. Plume measurement and modeling has shown 

that it is more than possible for larvae in the water column 1km away from a diffusion source to 

contact PW at 1% of it’s original concentration. 

The early life history stages of invertebrate larvae are very important developmental phases. 

Some invertebrates and algae have been shown to be particularly susceptible to any negative 

effects of contaminants during these developmental phases. The impacts seen on larval behavior 

have included swimming behavior, cue-recognition, settlement and larval survival. Little work 

has been published that follows the sub-lethal impacts on larvae through to their adult phase. 

The ecological consequence of carrying-over impacts to the adult phase from sub-lethal impacts 

on larvae have been rarely tested. 

OBJECTIVES: One of the major aims of this project was to test the assumption that sub-lethal 

impacts on larvae can, and do, carry-over into the adult phase of the invertebrate life-cycle. By 

exposing invertebrate larvae at an early developmental stage and following their development 

through to adulthood and beyond, it is possible track the impact of early exposure to PW. 

Other aims were more broad: to expand the number of larvae exposed to, and types of sub-lethal 

impacts assessed from exposure to PW. 

DESCRIPTION: By exposing different types of larvae to PW at various stages of their 

development, we had a number of outcomes: 

3⁄4 First, we expanded the number and type of larvae tested for sub-impacts from pulsed 

exposure to PW. 

3⁄4 Second, we tested the types of sub-lethal impacts that can occur on invertebrate larvae from 

exposure to PW. By doing so, we tested for the impact of PW on a number of sub-lethal 

endpoints, including swimming behavior, cue-recognition, attachment and metamorphosis. 

3⁄4 Finally, we followed some species of larvae through their development into adults and tested 

for any carry-over impacts for a range of endpoints, including growth, competitive ability, 

reproduction and survival. 

The project tested a wide range of invertebrate larvae, including the bryozoans Watersipora spp, 

Bugula neritina, Schizoporella unicornis, the red abalone Haliotis rufescens, the sea star Asterina 

miniata and the ascidian Botrylloides spp. for carry-over effects from the sub-lethal impact of 

exposure to produced water as larvae. This broad range represents four different phyla of both 
Effects of Produced Water on Complex Behavior Traits of Invertebrate Larvae 

3

introduced and local species as well as a broad range of types of larval development. All 

exposures were short-term, spiked exposure in still water conditions in the lab. 

SIGNIFICANT CONCLUSIONS: 

• All exposures were short-term, spiked exposure in still water conditions in the lab. 

• Not all experiments on all invertebrate larvae were successful. 

• Those experiments that were successful showed, in broad terms, that there was little evidence 

for strong sub-lethal effects on the growth, competitive ability or reproductive output of those 

invertebrates successfully studied. However sub-lethal impacts did occur. 

• Where mortality occurred, it tended to be larger in colonies of bryozoans in treatments 

exposed to concentrations of 10% PW. 

• Many and varied sub-lethal impacts were found from exposing larvae to a range of PW 

concentrations (with 10 % PW having the greatest and most consistent impact). 

• Sub-lethal impacts include decreases in swimming capacity, slowed metamorphosis, altered 

attachment and settlement behaviors, and delayed opercula development. 

• Not all invertebrate larvae showed all impacts, nor were tested for the full range of endpoints. 

• There was little evidence for strong carry-over effects to adulthood of larval PW exposure, 

with some caveats. 

STUDY RESULTS: The results of this study are fully contained within the Report “Effects of 

Produced Water on Complex Behavioral Traits of Invertebrate Larvae” presented to MMS.

STUDY PRODUCTS: A number of presentations (6) were given during this work as both 

invited and conference seminars. Three posters were also given at a range of conferences. The 

details of all these were contained in the annual reports written for MMS. 

There are three manuscripts in preparation from work down during this project: 

Boxshall, AJ and PT Raimondi, The sub-lethal impact on Watersipora subtorquata adults 

from exposing larvae to a toxicant. In prep. A 

Boxshall, AJ, and PT Raimondi, Carry-over effects on adults from exposing Bugula 

neritina and Schizoporella unicornis larvae to sub-lethal toxicant. In prep. B 

Boxshall, AJ and PT Raimondi, Impacts on adult Phragmatapoma californica of 

exposing larvae to a sub-lethal toxicant. In prep. C 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 4

FINAL STUDY REPORT 

Introduction 

Produced Water (PW) is a by-product of oil drilling and in the Southern California region a 

common form of disposal of PW is by direct discharge into the ocean from a platform-based 

diffuser. The actual content of PW is very variable on both the spatial (regionally, locally) and 

temporal (daily from the same field) scales. The salinity of many PWs in California is generally 

around the mid- to high-20's ppt. PWs can contain a large number of different compounds and 

elements, including polycylic aromatic hydrocarbons (PAH - e.g., benzene, toluene, naphthalene, 

phenols), metals (e.g., As, Cr, Ni, Ag, Cd, Cu, Pb, Se, Ba), and other compounds (e.g., cyanides 

and ammonia). Many of these compounds and elements are known to have toxic effects on a 

number of organisms however these toxic effects are not always lethal, even in static conditions 

(e.g., Ray and Engelhardt 1993). 

If larvae are entrained in a low concentration plume of PW, exposure could be on-going. 

However, plume dilution models suggest that many invertebrate larvae are likely to undergo 

spiked, rather than on-going, exposure to PW (e.g., Washburn et al 1999). A short, spiked 

exposure sees larvae exposed to a pulse of PW for a short period of time. This form of exposure 

is unlikely to produce lethal effects, however it may result in a series of important sub-lethal 

effects for organisms in various stages of their lifecycles (Raimondi and Schmitt 1993, Reed and 

Lewis 1994). 

Field studies in the early 1990s in the southern California Bight challenged the belief that the 

discharge of produced water from oil drilling platforms had little or no effect on organisms in the 

water column (e.g., Raimondi and Schmitt 1993, Krause et al 1993, Reed and Lewis 1994, Reed 

et al. 1994). This belief was based, in part, on the idea that the harmful components of PW have 

relatively short residence times in the water column. Based on acute (lethal) laboratory tests, the 

water-soluble contaminants in PW are believed to be diluted rapidly to levels well below those 

suspected to cause meaningful biological responses. However, the results of the studies noted 

above support the idea that the discharge of produced water can cause severe, generally sublethal, effects to organisms over distances well beyond that predicted by plume dilution models. 

Washburn et al (1999) modeled and measured the movement of diffused PW in the Santa 

Barbara channel. They found that the minimum initial dilution from the diffuser was 

approximately 100 times in summer in a zone within approximately 80m of the diffuser source. 

The dilution rate increased in winter to approximately 500 times. However, from modeling work 

they also found it was possible to get patches of PW up to 1000m from a diffusion source with 

time-averaged dilution factors of 100 and 1000 for summer and winter (respectively). Hence, it 

is more than possible that larvae in the water column even 1km away from a diffusion source 

could contact PW at 1% of it’s original concentration. 

The early life history stages of invertebrate larvae are very important developmental phases. 

Some invertebrates and algae have been shown to be particularly susceptible to any negative 

effects of contaminants during these developmental phases (Capuzzo 1987, Raimondi and Reed 
Final Study Report – Raimondi and Boxshall 

 5

1995). Bryozoan (Raimondi et al 1997) echinoid (Krause et al. 1993) and molluscan larvae 

(Raimondi and Schmitt 1993), and even algal spores (Reed and Lewis 1994) that are 

contaminated during this developmental phase show behavioral effects later in the larval phase. 

The impacts seen on larval behavior have included swimming behavior, cue-recognition, 

settlement and larval survival. Little work has been published that follows the sub-lethal impacts 

on larvae through to their adult phase. There is a suggestion that the metamorphosis that occurs 

between the invertebrate larval and adult phases can be thought of as “a new beginning” (e.g., 

Pechenik 1999, Pechenik et al, 2001). Clearly, if this is the case, sub-lethal impacts thought to 

be important for larvae may not be as important from the perspective of the adult organisms. 

Which the carry-over of impacts to the adult phase, the ecological consequence of sub-lethal 

impacts on larvae may be diminished. This question has been rarely tested. 

One of the major aims of this project has been to test the assumption that sub-lethal impacts on 

larvae can, and do, carry-over into the adult phase of the invertebrate life-cycle. By exposing 

invertebrate larvae at an early developmental stage and following their development through to 

adulthood and beyond, it is possible track the impact of early exposure to PW. 

By exposing different types of larvae to PW at various stages of their development, we tested a 

number of outcomes. 

3⁄4 First, we expanded the number and type of larvae tested for sub-impacts from pulsed 

exposure to PW. 

3⁄4 Second, we tested the types of sub-lethal impacts that can occur on invertebrate larvae from 

exposure to PW. By doing so, we tested for the impact of PW on a number of sub-lethal 

endpoints, including swimming behavior, cue-recognition, attachment and metamorphosis. 

3⁄4 Finally, we followed some species of larvae through their development into adults and tested 

for any carry-over impacts for a range of endpoints, including growth, competitive ability, 

reproduction and survival. 

By following invertebrates from release through metamorphosis to adulthood as a competing 

member of a marine assemblage, we can measure the impact of early PW exposure on a different 

component of the invertebrate’s growth or interactions. Despite surviving the initial exposure to 

PW, the ecological impact of PW exposure on both the organism itself and surrounding 

organisms is relatively unknown (e.g., see Schüürmann and Market 1997). 

In this project, we tried to use a wide range of invertebrate larvae, including the bryozoans 

Watersipora spp, Bugula neritina, Schizoporella unicornis, the red abalone Haliotis rufescens, 

the sea star Asterina miniata and the ascidian Botrylloides spp (see Methods for details on each). 

This broad range represents four different phyla of both introduced and local species as well as a 

broad range of types of larval development. Not all tests listed above were done with each 

invertebrate. 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 6

Methods 

General Comments 

In general, we did similar experiments on various invertebrate larvae. Details of the invertebrates 

are below (see “Organisms”) where a broad overview of the culturing techniques and origins of 

the various animals is presented. 

In all cases, all larval exposure to PW was short between 45 and 90 minutes and occurred in the 

laboratory at the Long Marine Laboratory (LML) at the University of California, Santa Cruz. 

Larval culturing facilities were available at LML, which included constant temperature, cultured 

algal food and flow through water systems. When larvae culture was required, two methods 

were used. A sunken, constant-flow method (similar to that for molluscs discussed in 

Strathmann, 1987) was used as well as raising small batches (>1 larva/ml) of larvae in ≥1l 

beakers of 0.2 μl filtered seawater. Batches were cleaned and fed cultured algae of various 

species every other day. 

In the following section, details of each experimental are discussed. At many times the same 

method was used for different larvae. This has been noted where it occurred. 

Produced Water 

The produced water (PW) used in this project was supplied by the Minerals Management Service 

(MMS) in California. We did not analyze the exact composition of the PW we used in these 

experiments. Under our agreement with the MMS, we do not know the exact origin of the 

platform/s from which the PW was taken. We do know there were two samples collected from 

different platforms on different days in southern California, most likely from within or near the 

Santa Barbara channel. There was one collection in 1997 and another in 1999. Most 

experiments used the 1997 collection. After collection of the PW, it was stored on ice. In the 

laboratory, it was frozen in small aliquots and stored at –80°C within 24 hours of collection. 

The PW used is not representative of all PW, or even all southern Californian PW (see papers in 

Ray and Engelhardt, 1993 for discussions of the variation in PW composition). The lack of 

variation in the samples of PW is a potential a source of experimental error. 

In all cases, we exposed the larvae to various concentrations of PW ranging from 0% to 10% 

(and 25% in some pilot tests) of pre-diffuser levels. Note that all PW concentrations are 

expressed in % of the pre-release concentrations of PW (i.e., as obtained from the platform 

downstream of the WEMCO) prior to release via a diffuser. PW was diluted in all experiments 

using 0.2 μm filtered seawater (0.2SW). The 10% PW treatment was included as a positive 

control as pilot studies indicated we could expect a sub-lethal larval response to this treatment. 

We did not expect this concentration to be lethal, however we believed it would elicit negative 

behavioral responses, such as altering swimming and settlement behaviors. For the same reason 

we included the 25% PW treatment in some pilot studies. A 10% treatment is quite a high 

concentration of PW and would generally only be found in close proximity to the diffuser array 
Final Study Report – Raimondi and Boxshall 

 7

of an oil platform (< ~10 m; pers. comm. Bill Ford, Chevron, 1997). Washburn et al (1999) 

modeled the movement of diffused PW in the Santa Barbara channel near where we understand 

the test PW was collected. They showed that the minimum initial dilution was about 100 times 

(i.e., 1% concentration of the raw PW) in summer (about 500 times in winter) in a zone within 

approximately 80m of the diffuser source. Hence, the 10% treatment should only be present 

quite close to the diffuser. A 25% concentration would be unlikely more than 1m from the 

diffuser array and was only used in pilot studies to produce a known larval response. Larvae are 

extremely unlikely to encounter PW at 25% in the water column. 

Organisms 

The bryozoan Watersipora subtorquata, is now a common introduced fouling organism in 

sheltered subtidal waters along the California coast (e.g., Rees 2000). Larvae of W. subtorquata

were used for a large proportion of this project. The bryozoan Schizoporella unicornis is a 

common native species found in sheltered subtidal areas along the Californian coast (Ricketts et 

al 1985). Both bryozoans brood larvae, which are released after exposure to light. Both have an 

encrusting, clonal growth form, making them a good target species to follow through settlement 

and subsequent growth as an adult. 

Larvae of W. subtorquata and S. unicornis were individually collected and stored in a communal 

beaker until exposed to PW within the first 2 hours of release. For all experiments using these 

bryozoans, the larvae came from multiple unrelated colonies collected from at least 3 different 

sites within the Santa Cruz Harbor, Santa Cruz, California, USA. At least 40 different adult 

colony fragments were used per experiment for both bryozoans. The adult W. subtorquata used 

for spawning in different experiments were from very different stocks and from three different 

seasons, thus reducing the chance we were re-testing the progeny of the same adults three times. 

S. unicornis were collected for a single experiment. 

The upright bryozoan Bugula neritina is ubiquitous across the globe (Keough 1989) and found 

commonly in bays, harbors and sloughs in California. Larvae of B. neritina are brooded and 

released from adults after exposure to bright light. Larvae were individually collected and stored 

in a communal beaker until exposed to PW within the first 2 hours of release. Adults used in 

these experiments were collected from Elkhorn Slough, Monterey Bay for use in both 1998 and 

1999. 

The colonial ascidian Botrylloides spp broods larvae which can be released after exposure to 

light. The adults are encrusting and common in sheltered subtidal areas of the California coast 

(Ricketts et al 1985). A similar protocol to that used for collecting the W. subtorquata and S. 

unicornis adults was used with Botrylloides spp. The adults used came from the Santa Cruz 

Harbor. We attempted experiments with the Botrylloides spp larvae twice in the summer of 1998 

and once in 1999 with limited success. 

The sea star Asterina miniata is common along the Californian coastline in rocky and sandy areas 

from the intertidal to >250 m (Ricketts et al 1985). We did a series of pilot experiments with the 

sea star in late 1997. These larvae were cultured using the standard 1-methyladenine method (see 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 8

Strathmann 1987 for details). Multiple adults used in these experiments were collected from the 

intertidal around Santa Cruz and raised in aquaria at LML, UCSC. 

The red abalone Haliotis rufescens is found subtidally in California. Culturing techniques are 

well-established (see Boxshall 2000 for information). H. rufescens larvae do not feed and are in 

the plankton for 7 days before they are competent to settle. They cue to a peptide associated with 

the phycobillins in coralline red algal species, which is a mimetic of the neurotransmitter, GABA 

(Morse and Morse, 1984). 

Specific Methods 

Watersipora subtorquata

We ran three experiments with similar methods (see Boxshall and Raimondi, in prep. A). The 

methods used for Watersipora subtorquata are a template for the methods used for other larvae. 

Differences will be noted when they occur. 

Larvae were exposed for between 50 and 65 minutes to PW of four concentrations (0%, 0.1%, 

1% and 10%). We grew the settlers in the lab for between 8 and 12 days and transferred them to 

the field for monitoring for between 40 and 150 days. For the first two experiments we pooled 

all larvae within a treatment into one beaker for the short duration of exposure. This is not the 

most ideal situation and was forced onto us by low numbers of larvae. It can be argued this 

results in pseudo-replication, particularly as larval behavior can be quite variable (see any paper 

in McEdwards 1995). It can also be argued that this practice reduces variation in application of a 

potentially variable toxicant. Except for this 50 - 65 minute period, the larvae and subsequent 

adults were raised and monitored individually for the duration of the experiments. 

To test if this short-term pooling during exposure in the first two experiments resulted in the loss 

of important information on the variability of larval reactions to PW we artificially formed 

batches of larvae for the 3rd Experiment and in some experiments with other invertebrates. If 

exposing larvae in batches resulted in treatment effects that were different between in batches 

(i.e., a treatment x batch interaction), we need to be careful with interpretations of the first two 

experiments. There were very few batch x treatment effects across a range of endpoints (see 

Results for details). 

During Exposure 

All larvae were exposed in 100ml of 0.2μm filtered seawater (0.2 SW) and PW (at the required 

dilution) in 250ml plastic beakers (Markson Lab Supplies) in static water conditions in a water 

bath at 15 -18°C. All beakers were swirled every 10-15 minutes to discourage settlement during 

exposure. For the three main experiments, PW concentrations ranged between 0% (the Control) 

and 10% of the raw PW concentration (see Table 1 for details). In experiment 1, there were 5 

treatment levels, but in experiments 2 and 3, there were 4 treatment levels. We did a separate 

behavioral trial in which the highest concentration was 25%. During exposure, the salinity (30.9 

to 31.1 ppt), pH (6.6 to 6.8) and dissolved O2 (4.35 - 4.5 mg/l) in the different treatments was 

within the narrow range of those seen in the 0.2SW used as a control, however the pH and 

dissolved O2 was at the lower end of this range in the 10% PW treatments. 
Final Study Report – Raimondi and Boxshall 

 9

After Exposure 

After exposure, the larval were transferred into beakers containing a 105μm mesh. We used the 

sunken filter technique (Strathmann 1987) at all times so as not to expose larvae to air. We 

flushed all beakers at least twice with 0.2SW to remove traces of PW. Larvae were transferred 

from exposure in the same order in which they were added. After flushing, larvae from each 

beaker were carefully washed into 0.2SW in 15ml Petri dishes and stored. When all larvae had 

been flushed and transferred (generally a 10-15 minute process), they were transferred 

individually into growth beakers. 

Growth beakers were 10ml disposable plastic beakers (Fisher Scientific) filled with ~8ml of 

0.2SW and contained only one larva each. The larval behavior (see “Behavioral Endpoints”) 

was noted within the first hours and at a number of times while being grown in the lab. Larvae 

were fed a 1ml mixture of phytoplankton (Isochyris and Rhodomonas) after they settled and 

water was changed at least every other day. 

Field Outplanting and Monitoring 

At outplanting, the colonies were generally only the ancestrula plus some of the first zooid. We 

gently cut out the plastic beaker around the colonies and glued it a PVC back-board (sizes of the 

boards ranged up to 75cm x 36 cm depending on the space required in each experiment), which 

was hung from floating docks in the Santa Cruz Harbor. 

For experiments 2 and 3 and days 110 and 150 only of experiment 1 (see Table 1 for details), the 

outplanted adults were assessed using a camcorder (Sony Hi-8 TR 400: x12 optical zoom) with 

close up filters (total possible magnification ~ x20). The video images were captured in the lab 

and analyzed using NIH Image for area, number of zooids and perimeter of colony (NIH Image is 

a public domain program developed at the US National Institutes of Health and available at 

http://rsb.info.nib.gov/nih-image/). For all census times before day 110 in experiment 1, 

colonies were counted using a field microscope. No data about colony size were taken at these 

census times. 

Table 1. A summary of the experimental conditions for W. subtorquata

Experiment, Start 

& Exposure Batches

Exposure 

Duration

Concentrations Outplanted Census 

Days

Larvae

1: 

November 1997 

(No Batches)

50 mins 0%, 0.01%, 

0.1%, 1%, 10% 

After 8 Days 

For 150 Days

Day 10,20, 

30, 39, 81, 

110, 150

89 total 

70 used 

2:

February 1998 

(No Batches)

50 mins 0%, 0.1%, 1%, 

10%

After 8 Days 

For 80 Days

Day 20, 25 

40, 60, 80

46 total 

39 used 

3:

August 1998 

(4 Batches)

65 mins 0%, 0.1%, 1%, 

10%

After 12 Days 

For 40 Days

Day 10, 20, 

40

323 total 

192 used 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 10

Behavioral Endpoints 

At each stage, we measured mortality and any visible abnormalities in the larvae or adults. 

Following are the endpoints for each phase of the experiments with W. subtorquata but many are 

common to the experiments with other larvae. 

During Exposure

3⁄4 The swimming behaviour of the larvae. This was assessed as either swimming or not. Other 

experiments ran parallel to the exposure experiments to quantify larval swimming (see 

“Behaviour Trial”). 

3⁄4 The number of larvae settled. 

After Exposure

In this phase, larval behavior was placed into one of 7 categories: 

3⁄4 Swimming; 

3⁄4 Searching - temporarily attached; 

3⁄4 Not moving; 

3⁄4 Metamorphosed; 

3⁄4 Operculum visible (in later counts, this became the number of opercula visible); 

3⁄4 Dead (clearly dead with a evidence remaining); 

3⁄4 and unknown (this included larvae that disappeared). 

Some of the categories were pooled for analysis (e.g., the categories: ‘searching - temporarily 

attached’ and ‘metamorphosed’ are both a part of the settlement process and were often lumped 

together as ‘settling’. 

We measured: 

3⁄4 Larval behaviour soon after exposure (within <1 to 3 hours). 

3⁄4 Larval behaviour one day (24 hours) after exposure. 

3⁄4 Larval behaviour at various times during the lab growth phase (details are shown where data 

are reported). 

Field Outplanting and Monitoring

The colony size was measured as both the number of zooids and zooid size (mm2). We measured 

a number of endpoints. 

3⁄4 The number of zooids at outplanting. 

3⁄4 The adult growth was measured as the change in the number of zooids between census dates 

(see Table 1 for census details). 

3⁄4 The zooid size (average zooid size in per mm2) was also calculated for each census date. 

This measure of colony size allows the zooids counts to be scaled for differential growth in 

the colonies by taking into account the area the colony occupies. 

3⁄4 The competitive ability of the colonies. This experiment was only attempted with 

Watersipora subtorquata. The competitive load on the colonies was assessed for each 

treatment. Competitive ability was measured as the ability of colonies to maintain 

themselves against competing neighbours (“draws”), the inability to withstand overgrowth by 

neighbours (“losses”), or the ability to overgrow neighbours (“wins”). A drawn situation is a 
Final Study Report – Raimondi and Boxshall 

 11

stalemate where neither the target colony nor the neighbour has the upper hand and there is a 

change in growth pattern of both competitors. A loss is when the target colony has been 

overgrown, whereas in a win the target colony has overgrown its neighbours. From a W. 

subtorquata colony perspective, these situations are not independent interactions as most W. 

subtorquata colonies will experience at least two of these conditions at one time. A loss 

results in the reduction of biomass and hence potential reproductive output due to 

overgrowth, and should be more detrimental to a colony than a draw. In a loss situation, we 

estimated the proportion of total area overgrown on the target colony. This was done on the 

computer screen with captured images by predicting the growth pattern in the absence of the 

ascidian based on the growth at this and previous census dates. It is an estimate as it is hard 

to define exactly where a colony is underneath a competitor. However, due to the controlled 

nature of the surface in this experiment, even after 150 days the colonies were quite regularly 

shaped. The competitors that were in large enough numbers to analyse were colonial 

ascidians of two genera: Botryllus and Botrylliodes. 

Analyses 

Mortality and behavior

To analyze most behaviors and mortality, we used hierarchical, log linear modeling with batch, 

PW concentration (treatment) and the behavior as categorical variables (Sokal and Rohlf 1995). 

Initially, a model is fitted with all the interaction terms and used to calculate the G2 statistic. To 

find the significance of each term, you remove that term, re-run the model and calculate a ∆G2

, 

which is compared to a distribution similar to the χ2 distribution. 

In Experiment 3, we exposed larvae to PW in four artificially allotted batches as opposed to the 

pooled exposure in experiments 1 and 2. We tested for an effect of batch on larval reactions to 

the treatments at various stages: 

3⁄4 The swimming behaviour of the larvae 1 hour after being removed from PW. 

3⁄4 The larval metamorphosis 24 hours after being removed from PW. 

3⁄4 The mortality 24 hours after being removed from PW. 

3⁄4 The number of colonies with opercula by Day 4. 

3⁄4 The mortality at outplanting. 

3⁄4 The size of colonies at outplanting. 

3⁄4 Mortality of the colonies at Day 10, Day 20 and Day 40. 

3⁄4 the size of the colonies at Day 10, Day 20 and Day 40. 

Of particular interested are any batch x treatment effects on larval behaviors: swimming, 

settled/metamorphosed, operculum development and survival. Some of the interactions are 

biologically meaningless and so we have not included them in the results. We have detailed the 

results for the batch x treatment x “behavior”, treatment x “behavior” and batch x “behavior”. If 

there is a significant batch x “behavior” interaction, it simply shows that larvae in different 

batches had different behaviors, regardless of treatment. This interaction may be biologically 

interesting but is not important in the context of these experiments as we are only interested in 

interactions of behaviors with the treatments. A significant batch x treatment x “behavior” 

indicates that the difference in behavior with treatment depends on the batch of larvae used. 

When there was a batch x treatment x ‘behaviour’ effect, we checked the frequencies for a 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 12

pattern. If there was a clear pattern, we removed that batch and re-ran the analysis. As this is 

technically an unplanned comparison (Sokal and Rohlf, 1995), we corrected the alpha level for 

the unplanned test. Generally there was only one extra test and hence we I used an error rate of 

α= 0.025. This is a very conservative error correction (Sokal and Rohlf, 1995). 

A significant treatment x “behavior” indicates that the larval behavior differs between treatments. 

To ascertain which treatments were important, we made 3 planned comparisons using the 

Fisher’s Exact χ2 or the Yate’s corrected χ2, whichever was appropriate (Sokal and Rohlf, 

1995). 

Growth and competitive ability

All data measuring growth and competitive ability of adults or larvae were analyzed using 

variations of ANOVA. Where necessary the data were transformed to maintain homogeneity of 

variances and normality (Underwood 1997). We analyzed the growth data with a repeated 

measures ANOVA where growth was the repeated measure (assessed as the change in number of 

zooids between census dates). 

The data in experiments 1 and 2 were analyzed using ANOVA with Treatment (fixed factor; 4 or 

5 levels) as the single factor. The data in Experiment 3 were analyzed using 2-way ANOVA 

with Batch (4 levels) and Treatment (4 levels) as fixed factors. The treatment effects in this 

experiment were compared between batches first and if no Batch x Treatment interaction was 

found, the data were pooled across batch for further analysis of the treatment effect. For each 

significant treatment effect, we compared the control to each treatment level in a pairwise 

comparison using a two-sided Dunnett test (Underwood 1997) as this was the most biologically 

interesting. Some marginally non-significant results with low power were also tested using this 

method. 

Due to low sample sizes in some experiments possibly resulting in increased Type II errors, we 

checked the power of all non-significant tests to pick up a change using Pass 6.0 

(www.ncss.com/pass). We based the effect size on natural levels of variation in this system and 

the alpha level was set at 0.05. 

Behavioral Trial

We ran one experiment separately from the exposure-outplant experiments to detail the behavior 

of the larvae during exposure. The larvae used in the behavior trial came from a subset of the 

batch of adults used in Experiment 3 and the trial started the day before the release of larvae for 

Experiment 3. 

We did the behavior trial in 20 ml disposable plastic beakers (Fisher Scientific) filled with at 

least 10 ml of 0.2SW and PW. In this trial, there were 5 treatments: 0%PW, 0.1%PW, 1%PW, 

10%PW and 25%PW with 5 replicates of each. We used 186 larvae placing multiple larvae in 

each beaker (4-8 larvae per beaker) and calculated the % swimming after 15, 30, 45, 75 minutes 

and 23 hours. We also noted if any larvae attached or metamorphosed. These larvae were never 

washed from the PW and never outplanted. These data did not require analysis for the swimming 

behaviors at 15 and 75 minutes, however we used a 1-way ANOVA to analyze the proportion 
Final Study Report – Raimondi and Boxshall 

 13

attached after 75 minutes and a two-sided Dunnett test to compare between treatments. After 23 

hours of constant exposure, we analyzed the proportion of larvae metamorphosed in each 

treatment with a similar one-way ANOVA and two-sided Dunnett test. 

For extra behavioral information we also quantified the swimming behavior of the larvae used in 

Experiment 3 during the first 10 minutes of exposure and up to 75 minutes of being transferred 

to clean 0.2SW. There were 4 replicate batches of each treatment in this test with between 14 

and 25 larvae in each. The analysis was the same as the main behavioral trial but data were 

analyzed for the first 5 to 10 minutes during exposure and at 75 minutes after exposure only. 

Schizoporella unicornis

We ran one experiment in June 1998 with methods similar to those used for Watersipora

subtorquata. The larvae were raised in the lab for 17 days after exposure to the 4 standard PW 

concentrations (Boxshall and Raimondi, in prep. B). Larval development was followed in 

parallel studies with similar behavioral endpoints measured as with W. subtorquata. The colony 

size was measured at outplanting. There was extraordinary growth on the outplanted boards for 

this experiment, which obscured much further analysis. Analyses were the same as those used 

above. 

Bugula neritina 

We ran a series of experiments in late 1999 using the larvae of the upright bryozoan, Bugula 

neritina (Boxshall and Raimondi, in prep. B). The general methods were very similar to those 

used for W. subtorquata. One important difference is in the method for assessing the size of the 

colony. As B. neritina have an upright growth form, the standard way to assess growth is to 

count the number of bifurcations in the colony (Keough 1989). The size of the colonies was 

measured at outplanting and at Day 70 when the experiment ended. Another difference was that 

the number of ovicells present on the colonies was counted at the end of the experiment. For 

analysis, the number of ovicells was scaled for size of the colony (i.e., number of bifurcations) 

and analyzed as log10 (ovicell density+1). This enables some estimate of reproductive 

differences between treatments. B. neritina were batched for all experiments. There were no 

differences between batches that affected the treatments (Boxshall, unpubl. data). 

Botrylloides spp.

We ran a series of experiments in mid 1998 using the larvae of the colonical ascidian, 

Botrylloides spp. The general methods were very similar to those used for W. subtorquata. 

These experiments were not successful as the larvae only swim for approximately 20 minutes 

before settling. We obtained preliminary data on the swimming behavior of Botrylloides spp 

(Boxshall, unpubl. data). 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 14

Asterina miniata 

The behaviors of these larvae to a biofilm cue were far less specific than we had hoped, making 

them untractable for use in PW experiments. Although an extensive series of pilot tests were 

complete, we were unable to find a tractable, cued surface to which this larvae would attach. 

Asterina miniata show very interesting swimming behaviors and would be very useful for future 

work into the detailed impacts of PW on swimming behaviors of invertebrates. 

Haliotis rufescens 

The abalone larvae were tested for settlement ability as competent larvae after exposure to four 

treatments of PW in 0.2SW (0%, 0.01%, 0.1% and 10%) for one hour. As a cue to settlement, 

10-6 GABA was used. The proportion of larvae settled in 20ml disposable beakers was assessed 

and compared to control beakers without GABA. The proportion of larvae settled was tested 

using a two-way ANOVA (Treatment: 4 levels; Cue: 2 levels). Data did not require 

transformation. 

Results 

General Comments 

This results section is a summary of many of the important results from the project. Further 

detailed results and analyses are to be published in a series of papers (Boxshall and Raimondi, in 

prep A, B and C). 

Specific Results 

Watersipora subtorquata

Batched Exposure vs Pooled Exposure

In summary there was no effect of batching the larvae during exposure on larval activities very 

early in life (i.e., swimming (table 2), metamorphosis at 23 hours (table 3), survival in the first 24 

hours (out of 185 larvae, only one had clearly died and one other had disappeared from different 

batches) or later as adults (growth at outplanting, and both growth and survival at Days 10 and 40 

(table 4)). However, there was a difference b/n batches in the development of opercula (Day 4) 

in the lab and the survival of juveniles to outplanting (table 5 and 6). These are early stages of 

development, but after metamorphosis. 
Final Study Report – Raimondi and Boxshall 

 15

Table 2: W. subtorquata swimming behaviors. Swimming 75 minutes after being washed from PW. 

 G2 df p ∆G2 df p

batch x tmt x sw 9.2970 9 0.410 

(full model) 

treatment x sw 15.77 12 0.202 6.47 3 0.091 

batch x sw 17.47 12 0.133 8.17 3 0.043

Table 3: W. subtorquata larval metamorphosis 23 hours after being washed from PW. 

 G2 df p ∆G2 df p

batch x tmt x met 10.4019 9 0.319 

 (full model) 

treatment x met 26.38 12 0.010 15.98 3 0.001

batch x met 13.31 12 0.347 2.90 3 0.407 

0% Vs 0.1% (i.e., without 1% and 10%) 

 G2 df p ∆G2 df p

batch x tmt x met 1.375 3 0.771 

(full model) 

treatment x met 10.18 4 0.037 8.81 1 0.003

batch x met 5.33 6 0.502 3.96 3 0.266 

 

0% Vs 10% (i.e., without 0.1% and 1%) 

 G2 df p ∆G2 df p

batch x tmt x met 4.142 3 0.247 

(full model) 

tmt x met 12.85 4 0.012 8.71 1 0.003

batch x met 4.46 6 0.614 0.32 3 0.956 

Table 4: Survival of W. subtorquata colonies to the final day in Experiment 3 (Day 40). 

 G2 df p ∆G2 df p

batch x tmt x surv 11.195 9 0.263 

(full model) 

tmt x surv 13.39 12 0.342 2.19 3 0.533 

batch x surv 11.58 12 0.480 0.39 3 0.943 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 16

Table 5: W. subtorquata survivorship to outplanting in Experiment 3, comparing the batches. We have included 

mean survival for comparison. 

% Total Survival

Treatment

Mean Batch 1 Batch 2 Batch 3 Batch 4

0% 98 92 100 100 100 

0.1% 95 100 100 100 80

1% 87 73 92 82 100

10% 84 92 83 92 67 

______________________________________________________________ 

Table 6: W. subtorquata survivorship to outplanting in Experiment 3.

 G2 df p ∆G2 df p

batch x tmt x surv 17.415 9 0.043

(full model) 

tmt x surv 24.06 12 0.020 6.64 3 0.084

batch x surv 19.32 12 0.081 1.91 3 0.592 

Re-analyzed without Batch 4. Note an alpha level of 0.025 this analysis. 

 G2 df p ∆G2 df p

batch x tmt x surv 6.478 6 0.371

(full model) 

tmt x surv 17.65 9 0.039 11.18 3 0.011

batch x surv 7.95 8 0.439 1.47 2 0.479

Re-analyzed survival as planned comparison s without Batch 4 to test which tmt differed from the 

control. 

 Pearson χ2

 df p Fisher’s Exact (p) 

0 % vs 10 % 1.934 1 0.354#

 *0.357 

0% vs 1% 8.016 1 0.013#

 *0.006

0% vs 0.1% ^ 

#

 We have used a corrected alpha level of 0.017 to test the null hypotheses due to previous comparisons of these 

data. 

* Due to small number of frequencies in some cells, I have used the conservative Yate’s corrected χ2

 in the 1% and 

10% comparison (Sokal and Rohlf, 1981). I have also shown the Fisher’s Exact test p for comparison. 

^ There is no difference in survival between batches 1,2 and 3 for this treatment. 

______________________________________________________________ 
Final Study Report – Raimondi and Boxshall 

 17

Behaviour during and after exposure

The larvae consistently swam less (Figure 1), settled less (Table 7) and showed less movement of 

any kind under the influence of higher concentrations of PW (Boxshall, unpubl. data). Not 

surprisingly, the 25% PW impacted heavily on larvae (table 7). There was little difference in the 

activity of larvae within hours of exposure to PW if they were washed in clean, filtered seawater 

(Table 8). 

After 24 hours, most larvae ×± sd : 72 ± 18%) had metamorphosed and there was no difference 

between treatments (df= 3, G2

 = 4.96, p= 0.176). By day 3 (66 ± 12%) or day 4 (77 ± 8%) the 

number of colonies with opercula also did not differ between treatments (Day 3: df= 3, G2

 =1.49, 

p= 0.684; Day 4: df= 3, G2 = 0.86, p= 0.836). 

There was no difference between all the treatments for the number of colonies that had developed 

opercula by either 3 (× ± sd : 73 ± 7%) or 4 (86 ± 6%) days after exposure (Day 3: df= 4, G2

=1.42, p= 0.840; Day 4: df= 4, G2

 = 1.76, p= 0.779). 

______________________________________________________________ 

Table 7. 

A. The proportion of W. subtorquata larvae attached (settling) after 75 minutes of exposure. These data did not 

require transformation. 

Source df MS F-ratio p

Treatment 4 0.249 12.808 0.000

Error 20 0.019 

Two-sided Dunnett test 

Tmt Mean differences p

 from control

0.1% -0.018 0.999 

1% -0.159 0.245 

10% -0.449 0.000

25% -0.452 0.000

B. The proportion of W. subtorquata larvae metamorphosed after 23 hours of constant exposure. These data 

were arcsine transformed due to the high number of cells with a value of 100%. 

Source df MS F-ratio p

Tmt 4 1.207 12.722 0.000

Error 20 0.095 

Two-sided Dunnett test 

Tmt Mean differences p

 from control

0.1% -0.024 1.000 

1% 0.043 0.998 

10% -0.257 0.501 

25% -1.127 0.000

__________________________________________________ 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 18

Figure 1. Proportion of Watersipora subtorquata swimming during exposure to PW after 15 minutes and 75 

minutes. 

Table 8. The percentage of W. subtorquata larvae showing each behavior 3 and 31 hours after exposure. The 

numbers in brackets are the actual number of each larvae showing each behavior. 

Percent Post-exposure behavior

Percent 3 hours after exposure Percent 31 hours after exposure

Treatment

total 

larvae 

swim settlement 

activities 

not 

moving 

dead swim settlement 

activities 

not 

moving 

dead 

0% 10 10 (1) 90 (9) 100 (10) 

0.01% 15 80 (12) 7 (1) 13 (2) 87 (13) 13 (2) 

0.1% 15 13 (2) 80 (12) 7 (1) 87 (13) 13 (2) 

1% 15 7 (1) 73 (11) 20 (3) 93 (14) 7 (1) 

10% 15 67 (10) 20 (3) 13 (2) 80 (12) 20 (3) 

Mortality

When larval mortality occurred, it tended to be in higher concentrations of PW and was visible 

after the first hours of exposure rather than during exposure. There was no mortality during 

exposure in Experiments 1, 2 or 3. 
Final Study Report – Raimondi and Boxshall 

 19

In Experiment 1, there was no mortality at all in the control (0%) during the laboratory phase of 

this experiment. What mortality that did occur was in PW treatments. It was not possible to test 

these data due to small sample sizes (low mortality in >1/3 of the cells). However, there was 

little mortality and very little difference between treatments with PW at any census time in the 

lab. At outplanting, there had been a uniform 20% mortality in all the PW treatments. 

Most mortality in the field occurred between Day 39 and Day 81. However by day 81there was 

no difference in mortality between the treatments (df=4 , G2 =1.82 , p=0.768). By Day 150, there 

was no difference in mortality between the treatments (table 3; df=4 , G2 =2.04 , p=0.728) but 

note that the there was more mortality in the 10% treatment than in the 0%. The highest 

survivorship of colonies that were outplanted was in the 1% treatment. 

In Experiment 2, there was no difference in mortality between the treatments 15 minutes after 

exposure (only 3 larvae dead: 2 in 0.1%, 1 in 1%), 24 hours after exposure (total of 4 larvae 

dead: 3 in 0.1%, 1 in 1%) or 8 days after exposure, prior to outplanting (df= 3, G2

 = 1.27, p= 

0.736). The small numbers in this experiment meant that testing for mortality differences was 

generally not possible (low mortality in >1/3 of the cells). There was little difference in mortality 

as the colonies grew in the field. The only sizeable mortality occurred in the 10% treatment and 

mainly between Day 40 and Day 60. In experiment 2, the highest mortality was in the 10% 

treatment and the highest survivorship was the 1% treatment, as in experiment 1. 

In experiment 3, there was little early mortality in this experiment in the lab. There was no 

mortality within an hour of transferring the larvae from PW to clean 0.2SW. The mortality 24 

hours after being removed from PW was very low. Two larvae out of 185 were dead or missing. 

There was no batch x treatment effect on early mortality. 

There was increased mortality at outplanting in treatments with higher PW concentrations. 

However, there was no clear pattern amongst batches. There was a significant batch x treatment 

x survival interaction but the reasons are not that clear (Table 6). Batch 4 seemed to be driving 

that result, as evidenced by the lack of batch x treatment x survival interaction in the analysis 

without batch 4 (table 6). When batches 1,2 and 3 were pooled and re-analyzed, there was no 

statistical difference in survival between the control and 10 % treatment, however there was 

statistically greater mortality in the 1% treatment than in the control (table 6). 

There was very little difference in mortality of the colonies at day 10 or day 20, between the 

treatments or within the batches in each treatment and no statistical differences at day 40. At 

days 10 and 20 it was not possible to test for a batch x treatment interaction, or even treatment 

effects as not enough larvae had died. Only 8 larvae out of 128 were dead at day 10 (15/128 at 

day 20). There was little clear pattern to this slight mortality, but the mortality in the 10% 

treatment was always greater than the control. Most mortality occurred from day 20 to day 40. 

When the mortality from day 20 to day 40 was compared, there was no difference in survival 

between treatments or within batches (Table 9). 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 20

Table 9. Experiment 3. W. subtorquata survival from Day 20 to Day 40.

 G2 df p ∆G2 df p

batch x tmt x surv 14.460 9 0.107 

(full model) 

tmt x surv 16.22 12 0.181 1.76 3 0.624 

batch x surv 14.55 12 0.267 0.09 3 0.993 

Growth

In experiment 1, the colonies were the same size in all treatments at outplanting (F4,53 =1.040, 

p=0.396), but given the variability in the sizes, the power was low (26%). From outplanting to 

day 150, the growth trajectory was similar between all treatments (figure 2, Table 10). There 

was no effect of PW exposure at all on the growth rates over time. 

Table 10. Experiment 1. Repeated Measures of W. subtorquata growth rates from Day 0 to Day 150. Given the p 

values, I have only included the univariate results and given the epsilon values, we have included the GG corrected 

p-values. 

Between Subjects

Source df MS F-ratio p

Tmt 4 0.468 2.026 0.120 

Error 26 0.231 

Within Subjects

Source df MS F-ratio p G-G

Time 6 11.787 76.591 0.000 0.000 

Time*Tmt 24 0.086 0.557 0.953 0.804 

Error 156 0.154 

Greenhouse-Geisser Epsilon: 0.3261 

Huynh-Feldt Epsilon: 0.4066 

The final size of the colonies, measured simply as the number of zooids, was not different 

between treatments at day 150 (F4,30 = 1.320, p=0.285), however the power in this test was low 

(32%) and there is a trend for smaller final size in the 10 % PW. The average final size of the 

zooids at Day 150 was not different between treatments (table 11, figure 3). As the result was 

marginal at an =0.05 level (p=0.061) and the power was low (power=58%), we ran a pairwise 

comparison of the PW treatments with the control. There was no difference between any of the 

treatments and the control (table 19). Due a priori decisions, we only tested the PW 

concentrations against the control. Constrained by degrees of freedom, we can only speculate 

that the effects of exposure may be stronger at intermediate concentrations (Figure 3). 
Final Study Report – Raimondi and Boxshall 

 21

Table 11. The size of W. subtorquata zooids in Experiment 1 at day 150. These data did not require transformation. 

Source df MS F-ratio p

Treatment 4 0.572 2.541 0.061 

Error 29 0.225 

Two Sided Dunnett Test 

Tmt Mean differences p

 from control 

0.01% -0.277 0.696 

0.1% 0.439 0.292 

1% 0.288 0.598 

10% -0.106 0.984 

In experiment 2, all the colonies surviving to outplanting were the same size (F3,34 =0.242, 

p=0.867), as were the subset chosen to ouplant (F 3,22 =0.850, p=0.482). From outplanting to 

Day 80, there was no difference in the growth trajectory due to the treatments (table 12). The 

final size of the colonies (day 80), measured as the number of zooids ×± se : 521 ± 50) was not 

different between treatments (F 3,17 =0.321, p=0.810). Nor was the average size of the zooids (×

± se:0.617 ± 0.03 mm2) different between treatments at day 80 (F 3,22 =0.282, p=0.838). 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 22

Figure 2. Experiment 1. Adult growth in the field for W. subtorquata over the entire duration of the experiment. 

Growth is measured in Log10 (growth+1), where growth = the number of zooids added between census dates. T0 to 

T10 represent the different PW treatments and time is measured in days. 

0 40 80 120 160

TIME

0

1

2

3

T0

0 40 80 120 160

TIME

0

1

2

3

T001

0 40 80 120 160

TIME

0

1

2

3

T01

0 40 80 120 160

TIME

0

1

2

3

T1

0 40 80 120 160

TIME

0

1

2

3

T10
Final Study Report – Raimondi and Boxshall 

 23

In experiment 3, all colonies were statistically the same size (measured as the number of zooids) 

at outplanting. There was no interaction with, or difference due to the main effect of, batch when 

the colonies were outplanted (Table 13). Also colonies had a similar number of zooids 

regardless of treatment. 

From outplanting to Day 40, there was no difference in the growth trajectory for any treatment or 

due to any batch x treatment interactions (Table14). The average size of zooids at day 40 

differed between treatments. At the alpha = 0.05 level, there was no batch x treatment 

interaction, however there was a significant effect of treatment (Table 15). The test of batch x 

treatment (p=0.056) has a power of 100% so we are comfortable there was no batch x treatment 

effect at an alpha = 0.05 level. There is no consistent pattern amongst treatments. I pooled the 

batches and have analyzed the zooid size between the treatments (table 15). There is a statistical 

difference between some of the treatments however none are different to the control. 

Figure 3. Experiment 1. The final size of W. subtorquata colonies at Day 150. Both axes are displayed as logs. 

_____________________________________________________________ 

2468

1012

Treatment

0

1

2

3

4

Zooid Size at Day 150
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 24

Table 12. Growth rates of W. subtorquata throughout Experiment 2. The univariate results from a repeated 

measures ANOVA. These data have been log10 transformed. 

Within Subjects df MS F-ratio p G-G

Growth 4 10.815 505.340 0.000 0.000 

Growth x Tmt 12 0.022 1.021 0.445 0.439 

Error 48 0.021 

Greenhouse-Geisser ξ 0.608 

Huynh-Feldt ξ: 0.964 

Table 13. The size of W. subtorquata colonies in Experiment 3 at outplanting. 

Variable df MS F-ratio p

Batch 3 0.104 0.529 0.663 

Tmt 3 0.063 0.322 0.810 

Batch*Tmt 90.169 0.858 0.564 

Error 149 0.197 

Table 14. Growth rates of W. subtorquata throughout Experiment 3. This repeated measures reports the 

unadjusted p-values as the G-G and H-F ξ values are 1 or close to it . 

Within Subjects df MS F-ratio p

Growth 2 37.294 922.053 0.000

Growth x Batch 6 0.057 1.421 0.211 

Growth x Tmt 6 0.039 0.975 0.445 

Growth x Batch x Tmt 18 0.049 1.223 0.251 

Error 134 0.040 

Greenhouse-Geisser ξ: 0.982 

Huynh-Feldt ξ: 1.000 

_____________________________________________________________ 
Final Study Report – Raimondi and Boxshall 

 25

Table 15 

A. Experiment 3. Average W. subtorquata zooid size (log10(zooid size+1)) at the final census date (Day 40). This 

test has a power of 100%. 

Source df MS F-ratio p

Batch 3 0.001 1.518 0.217 

Tmt 3 0.006 5.843 0.001

Batch x Tmt 9 0.002 1.973 0.056*

Error 69 0.001 

B. Experiment 3. Average W. subtorquata zooid size (log10(zooid size+1)) at the final census date (Day 40) pooled 

across batches. 

Source df MS F-ratio p

Tmt 3 0.007 6.485 0.001

Error 81 0.001 

Two Sided Dunnett Test 

Tmt Mean differences p

 from control

0.1% -0.020 0.117 

1% 0.022 0.062 

10% 0.008 0.795 

Competitive ability 

In experiment 1, we could identify a total of 7 deaths in the entire experiment due directly to 

overgrowth. There was no real pattern, with each treatment represented once except for 3 from 

0.1%. At day 150, every colony except one was interacting on average with at least 3 colonial 

ascidians. There was no difference in the number of ascidians (×± se : 4.3 ± 0.4) interacting 

with target colonies across treatments (F4,31 = 0.744, p=0.569) although there was only 17.5% 

power. This measure can be considered a crude indicator of the overall competitive load on the 

colonies due to interactions with colonial ascidians. There were never situations where the W. 

subtorquata colony clearly overgrew the ascidian (a “win”). Almost every colony was involved 

in a “draw” with multiple ascidian competitors but there is no clear pattern from these data (F4,26

= 0.648, p=0.633; Figure 4). Most colonies had a “loss” but again there was no clear pattern 

across treatments (F4,13 = 1.263, p=0.334, power = 12%; Figure 4). There was no difference in 

the proportion of estimated area lost through overgrowth (F4,14 = 0.728, p=0.587) and, again, the 

power was low (17%). These data were log10 transformed to maintain normality for analysis. 

Of the 21 colonies remaining in experiment 2 at day 80, ten were interacting with neighboring 

colonial ascidians however there was no clear pattern from these interactions. Eleven colonies 

had no neighbors at the time of census, including all remaining colonies from the 10% treatment. 

No colonies were being overgrown by colonial ascidians (Loss), 3 were overgrowing colonial 

ascidians (Wins: 1 x 0%, 2 x 1%) and 7 had 1 or more colonial ascidians touching them without 

a clear overgrowth by either the bryozoan or ascidian (Draws). The only colonies that clearly 

died from overgrowth were both from the 10% treatment (1 at Day 40, 1 at Day 80). Overall, 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 26

again there was no clear decrease in competitive abilities across treatments. Due to the shorter 

duration of experiment 3, there were not enough competitive interactions for analysis. 

Figure 4. Competitive interactions of W. subtorquata with colonial ascidians. Note the difference in scales. For 

details of what constitutes ‘Losses’ and ‘Draws’, see text.

0 0.01 0.1 1 10

Treatment

1

2

3

4

5

LOSSES

0

1

2

3

4

5

DRAWS

0 0.01 0.1 1 10

Treatment
Final Study Report – Raimondi and Boxshall 

 27

Schizoporella unicornis

The Schizoporella unicornis larvae showed an interesting pattern similar to W. subtorquata 

larvae. After four minutes of exposure to PW, the trend was for larvae to be swimming more in 

the lower PW concentrations. Those not swimming were searching on the surface or not moving. 

More were in the “not moving” category at 10% PW concentration (Figure 5). After 74 minutes 

of exposure, there were more larvae swimming in the higher concentrations. However, at this 

time, those in the lower concentrations had started to attach and metamorphose. By 6 hours of 

exposure, many more S. unicornis larvae had metamorphosed in the control than any other PW 

treatment, especially when compared to 10% PW (figure 5). Mortality was negligible during this 

exposure test, and after exposure in other tests while larvae were in the laboratory. 

Figure 5. The proportion of S. unicornis larvae swimming and metamorphosed at different times after exposure to 

PW. Note the different scales. 

0.2

0.4

0.6

0.1

0.2

0.3

0.1

0.2

0.3

0.4

0.5

4 mins 74 mins

6 hours

0 0.1 1 10

Treatment

0 0.1 1 10

Treatment

0 0.1 1 10

Treatment

Proportion Metamorphosed

Proportion Swimming
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 28

By the time for outplanting the S. unicornis colonies, there was no difference in the size of the 

colonies as measured by the number of zooids, between the treatments. There was no difference 

between the four batches of larvae, however there was a trend for the colonies in 10% PW to be 

smaller than the other colonies (Table 16, Figure 6). 

___________________________________________________________ 

Table 16. The size of S.unicornis colonies at outplanting to the field. 

Source df MS F-ratio p

Batch 3 9.093 0.865 0.461 

Tmt 3 25.870 2.462 0.066 

Batch x Tmt 9 11.891 1.132 0.347 

Error 109 10.509 

_____________________________________________________________ 

Figure 6. The size of Schizoporella unicornis colonies at outplanting. 

0

5

10

Number of Zooids

0 0.1 1 10

Treatment
Final Study Report – Raimondi and Boxshall 

 29

Bugula neritina 

After 15 minutes of exposure to PW, almost all the larvae in the 0%, 0.1% and1% treatments 

were swimming, compared to <10% of larvae in the 10% PW treatment. After 1 hour and 40 

minutes of exposure, most larvae were still swimming in the 0.1% and1% treatments but most of 

the larvae in the control had metamorphosed. This was not the case for the 10% PW treatment. 

Most larvae were not moving (figure 7). Mortality was negligible while larvae were being 

exposed to PW. This results was mirrored in the parallel experiment which exposed larvae ready 

for outplanting of the adults. 

Figure 7. Proportion of Bugula neritina larvae swimming after 15 minutes and 1 hour 40 minutes of exposure to 

PW. The proportion of larvae attached at 1 hour and 40 minutes is also shown. Note the difference in scales. 

0.2

0.4

0.6

0.8

1.0

Proportion attached

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

1.0

15 mins 1 hr 40 min

1 hr 40 min

Proportion swimming

0 0.1 1 10

Treatment

0 0.1 1 10

Treatment

0 0.1 1 10

Treatment
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 30

By the time for outplanting the B. neritina colonies, there was no difference in the size of the 

colonies as measured by the number of zooids (not bifurcations), between the treatments (table 

17). On average colonies were 5.5 ± 0.14 (× ± SE) zooids in size. Despite a difference in the 

size of the batches of larvae, there was no interaction between treatment and batch. 

Table 17. The size of Bugula neritina colonies at outplanting to the field. 

Source df MS F-ratio p

Batch 3 11.510 4.574 0.005 

Tmt 3 0.280 0.111 0.953 

Batch x Tmt 9 2.248 0.894 0.533 

Error 119 2.516 

After 70 Days in the field, there was no difference in the size of the colonies as measured by the 

number of bifurcations (table 18, figure 8). 

Table 18. The size of Bugula neritina colonies after 70 days in the field. 

Source df MS F-ratio p

Tmt 3 2.634 0.790 0.505 

Error 58 3.336 
Final Study Report – Raimondi and Boxshall 

 31

Figure 8. The size of Bugula neritina colonies after 70 days in the field, measured by the number of bifurcations 

present on colonies. 

Interestingly there was no difference in the density of ovicells present on the colonies after 70 

days in the field (table 19, figure 9). The number of ovicells were scaled for the size of colonies 

(number of bifurcations). Not surprisingly, there was a great deal of variability in the number 

and density of ovicells (note figure 9 is on a log scale). Despite this variability, there is a trend to 

decreased reproductive output as PW concentration increases. We counted all ovicells (full and 

empty). 

Table 19. The size of Bugula neritina colonies after 70 days in the field. 

Source df MS F-ratio p

Tmt 3 4.429 0.875 0.460 

Error 58 5.064 

2468

1012

6

8

10

12

14

16

Number of bifurcations

Treatment
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 32

Figure 9. The density of ovicells present on Bugula neritina colonies after 70 days in the field, measured as 

Log10(ovicell density+1). 

Haliotis rufescens 

We did a small experiment using the larvae of Haliotis rufescens comparing the complex 

behavior of settlement cue recognition amongst larvae exposed to four levels of PW for one hour. 

Not surprisingly, proportionally more larvae settled when the cue (GABA) was used than in the 

control. After 32 hours had elapsed since exposure, there was no difference between the 

behavior of larvae in all PW treatments (table 20, figure 10). 

Table 20. The proportional settlement of abalone larvae in four PW treatments (05, 0.01%, 0.1% and 10%) with 

and without a settlement cue. These data did not require transformation.

Source df MS F-ratio p

Cue 1 1070.249 27.314 0.000 

PW 3 33.548 0.856 0.478 

Cue*PW3 74.645 1.905 0.157 

Error 23 39.183 

_____________________________________________________________ 

2468

1012

Treatment

0

1

2

3

4

5

6

7

Log10(ovicell density+1)
Final Study Report – Raimondi and Boxshall 

 33

Figure 10. The proportional settlement of abalone larvae in four PW treatments (05, 0.01%, 0.1% and 10%) with 

and without a settlement cue. 

60

70

80

0%

60

70

80

0.01%

control GABA

60

70

80

90

Proportion settled

0.1%

50

60

70

80

90

10%

Proportion settled

Cue

control GABA control GABA

Cue
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 34

Discussion 

Overall, this project found there was little evidence for strong sub-lethal effects that carried over 

from the larval phase and impacted the growth or competitive abilities of the subsequent 

Watersipora subtorquata adults (see also Boxshall and Raimondi, in prep. A). The studies of W. 

subtorquata were done three times across three different seasons. Each time, the results were the 

same. There is certainly evidence for (at times) quite strong sub-lethal impacts on the larvae of 

Watersipora subtorquata and the other larvae studied. Although not as extensively studied, the 

other invertebrates in this study showed similar patterns for the adults (see also Boxshall and 

Raimondi, in prep. B and C). Although this is an important finding for the protection of some 

planktonic organisms from impacts of the release of PW, there are a number of caveats. 

One important caveat is that although mortality was generally low and not statistically 

significant, as would be expected from tests deigned to have deliberately sub-lethal impact, there 

were small differences seen in mortality of adults from larvae exposed to different concentrations 

of PW. When mortality occurred, it tended to be larger for Watersipora subtorquata colonies in 

treatments with spiked exposure to concentrations of and above 10% pre-release levels. These 

may be biologically interesting. Marine assemblages are highly variable, where there can be 

large variations in natural mortality from many sources (e.g., Connell 1978). Such small changes 

in adult mortality due to PW could simply be swamped by the natural high variability of marine 

populations. This caveat should be addressed by further work, possibly modelling of impacts 

(see Forde et al. in press). 

Higher concentrations of PW affected larval behaviour of Watersipora subtorquata during 

exposure. Larvae generally swam less, settled less and showed less movement of any kind under 

the influence of higher concentrations of PW. This may be due to a narcotic effect from an 

unidentified constituent of PW. This speculation is based on the evidence that larvae placed into 

clean, filtered seawater immediately began to show a range of ‘normal’ behaviours. Generally 

there was little difference in the activity of the larvae within hours of exposure to PW, if they 

were washed in filtered seawater. Where there was larval mortality it tended to be in higher 

concentrations and was visible after the first hours of exposure. Although, as stated above, 

mortality in most of these experiments was low. 

One important question that arises from these data is that if PW does not greatly impact the 

growth rates of invertebrates as adults, what is the fate, in a field situation, of those larvae that 

have had their behaviours altered? It is important to note that the all the larvae in this study were 

exposed in a laboratory situation. In this benign environment, individual larvae could be studied, 

followed, cleaned of PW and continue to live. If larvae pass through a cloud of PW in the ocean, 

stop swimming and sink, do they start to swim again once out of the PW? Or is there another 

fate? Are they more susceptible to predators? What about delayed metamorphosis and 

development? Does this leave them open to other natural impacts in the field? These remain 

unanswered questions. 

Information from the outplanting of Schizoporella unicornis colonies after the exposure of larvae 

to PW was unfortunately truncated due to field logistics however there are some data being 
Final Study Report – Raimondi and Boxshall 

 35

reconstructed (Boxshall and Raimondi, in prep. B). At the time of outplanting there was little 

statistical difference in the size of S. unicornis colonies, although the pattern of increased size 

with increasing PW concentration was not continued through to the 10% PW concentration. 

Further study will be needed on the impact on subsequent adults to strengthen conclusions. 

There were very clear sub-lethal impacts on the larvae of S. unicornis that tracked increasing PW 

treatment quite well. The higher the concentration of PW, the less the swimming ability of newly 

released S. unicornis larvae. When these same larvae began to attach and metamorphose, the 

pattern of metamorphosis similarly tracked the PW concentration. 

Like the other two bryozoans, there were strong sub-lethal impacts of PW on the behavior of 

larvae of Bugula neritina. Also like the other bryozoans, the carry-over impacts from the larval 

phase to adulthood were not strong. In the case of Bugula neritina, we were able to track the 

adults for 70 days in the field and follow their growth as well as gain some insight into their 

reproductive capacity. It is important to note that this measure of reproductive output can only be 

viewed as a single, snap-shot impression of the reproductive output from Bugula neritina. If it 

would be possible logistically, a better estimate of reproductive output would be to count the lifetime output from adult colonies. From our single estimate of reproduction, there was no strong 

impact carried-over from larval exposure to different concentrations of PW. This result should 

be viewed as a precursor to more analysis. Very late in the period of this project, access was 

granted to new supplies of PW by MMS. With this access, it was possible to re-visit the question 

of carry-over impacts on reproductive success of B. neritina. As a consequence, more data was 

generated after the project time had concluded. This is being further analyzed and will be 

published in future (i.e., Boxshall and Raimondi, in prep. B). 

Given the sub-lethal impacts previously seen in field studies using the abalone Haliotis rufescens

(Raimondi and Schmitt 1993) it was surprising to note that the complex behavior of cue 

recognition by the abalone was not impacted by PW exposure. The swimming capacity of 

Haliotis rufescens larvae were impacted during other pilot tests (Boxshall, unpubl. data). 

On the topic of PW concentrations, as previously discussed, exposure to 10% PW concentrations 

for one hour is unrealistically high exposure based on current plume dilution studies. This 

concentration was deliberately chosen to elicit a behavioural response (which it did) and to allow 

us to follow these larvae/adults through life to look for long-term effects. The 25% PW was used 

for similar reasons but no adults of any species were tracked through life. One important source 

of error in this project was the origin of the PW stocks. As noted, PW used for experiments 

came from 2 collections on single days at single (unknown) platforms. If there is to be the 

capacity to generalise about the impacts of PW (per se) on the ecology of marine organisms, 

studies must be done using a range of PW from a range of sources. The composition of PW is 

known to be very variable (see multiple papers in both Ray and Engelhardt, 1993 and 

Schuurmann and Market, 1997). 

There were very few interactions with treatment produced by exposing the larvae to PW in 

batches or as one large pool. When larvae were not batched, except for this 50 - 65 minute 

period, the larvae and subsequent adults were raised and monitored individually for the duration 

of the experiment. Of course, the exposure is an important time. The practice of batching larvae 
Effects of Produced Water on Complex Behavioral Traits of Invertebrate Larvae 

 36

is in sharp contrast to common procedure in many LC50-96 tests where organisms are treated 

and monitored as pooled groups throughout the study. The behaviors of larvae are inherently 

variable (see any paper in McEdwards, 1995) and so this issue should not be dismissed. 

However, it should also be seen within the context of the previous discussion about the lack of 

replication in sources of PW. 

When effects from batching larval exposure were seen, they tended to occur in later in the early 

stages of young adult development, not in the early larval swimming, metamorphosis or survival 

in the first 24 hours. Batch effects were also generally not present later as adults (e.g., growth at 

outplanting, and both growth and survival later). However, there were batch effects in the 

development of opercula of the bryozoan W. subtorquata (Day 4) in the laboratory and the 

survival of juvenile W. subtorquata to outplanting. These are early stages of development, but 

after metamorphosis. This is an interesting pattern worth noting but for which we have no 

present speculative solutions. 

The competitive ability of W. subtorquata adults were assessed after 150 days in the field. This 

time was required for enough competitors to grow around the colonies. The overall competitive 

load was no different amongst colonies in different PW treatments. The competitors were mainly 

ascidians. No PW treatment showed a diminished ability to compete. The unfortunately low 

power in these tests is difficult to overcome due to the variable and unpredictable nature of 

settlement in the field. The design of this experiment is ultimately controlled by where and when 

competitors settle. 

This project was successful in addressing the question of carry-over impacts from exposed 

invertebrate larvae to the adult phase of their life-cycle. A number of important further questions 

were raised as a result of this study. During this project, various personnel were involved, 

gaining valuable learning experience. They are listed in Appendix 1. 
Final Study Report – Raimondi and Boxshall 

 37

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The Department of the Interior Mission 

As the Nation's principal conservation agency, the Department of the Interior has responsibility for most 

of our nationally owned public lands and natural resources. This includes fostering sound use of our 

land and water resources; protecting our fish, wildlife, and biological diversity; preserving the 

environmental and cultural values of our national parks and historical places; and providing for the 

enjoyment of life through outdoor recreation. The Department assesses our energy and mineral 

resources and works to ensure that their development is in the best interests of all our people by 

encouraging stewardship and citizen participation in their care. The Department also has a major 

responsibility for American Indian reservation communities and for people who live in island territories 

under U.S. administration. 

The Minerals Management Service Mission 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) primary 

responsibilities are to manage the mineral resources located on the Nation's Outer Continental Shelf 

(OCS), collect revenue from the Federal OCS and onshore Federal and Indian lands, and distribute 

those revenues. 

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program

administers the OCS competitive leasing program and oversees the safe and environmentally sound 

exploration and production of our Nation's offshore natural gas, oil and other mineral resources. The 

MMS Royalty Management Program meets its responsibilities by ensuring the efficient, timely and 

accurate collection and disbursement of revenue from mineral leasing and production due to Indian 

tribes and allottees, States and the U.S. Treasury. 

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially affected 

parties and (2) carrying out its programs with an emphasis on working to enhance the quality of life for 

all Americans by lending MMS assistance and expertise to economic development and environmental 

protection.