### 1 | Page (Version: 26th May 2009 )



## OECD DRAFT GUIDELINES FOR TESTING OF CHEMICALS

PROPOSAL FOR A NEW GUIDELINE

Harpacticoid Copepod Development and Reproduction Test with Amphiascus tenuiremis

INTRODUCTION

1. Since 1995, revision and development of Organisation for Economic Co

- operation and Development (OECD) Test Guidelines in aquatic ecotoxicity testing have assigned highest priority to the development of acute and chronic tests with species of marine Crustacea (1). Specific recommendations to

deve lop subchronic/chronic test methods with both warm and cold

- water marine and brackish water species have been made (2). This Guideline describes test procedures for measuring the (sub)chronic effects of chemicals on an estuarine/marine harpacticoid copepod species under semistatic conditions, and the biological endpoints for such test procedures include survival and development of the early life

- stages, and multiple aspects of its reproduction ( e.g. , intersexuality, mating success, egg

- clutch size, abortion

rates, necrotic and infertile eggs, and numbers of viable offspring produced).

2. Copepods occur widely in marine, brackish and freshwater ecosystems. They represent important prey items for the larvae of many fish and larger invertebrates (3), and they a re increasingly used as a live food source in aquaculture (4). A number of copepod species (predominantly marine and brackish water species) have been used for many years to evaluate the acute and chronic toxicity of single chemicals and complex mixtures. For assessment of marine contaminants in temperate ecosystems, Amphiascus

tenuiremis

, Nitocra spinipes , and Tisbe battagliai have e.g. proven to be valuable test organisms (5

- 11).

3. The biological attributes of harpacticoid copepods facilitate the measur ement of development, reproductive and life

- table parameters, which provide sensitive and ecologically relevant endpoints that can be used to determine the potential (sub)chronic toxicity of many water

- borne contaminants.

4. Definitions used are given in A nnex 1.

PRINCIPLE OF TEST



### 5. Newly hatched larvae (termed nauplii), aged less than 24 hours at the start of the test, are exposed individually (60

- 120 nauplii allocated over 3 replicate microplates per treatment) in microwell (300

- μ L

total volume ) test ch ambers to seawater ( i.e. , control) and to the test substance added to seawater at a range of concentrations. The test duration is approximately 36 days at 25oC, which is sufficient time for the control animals to reach adulthood, mate and produce at least two clutch es of offspring (termed the F 1

### 2 | Page generation). The duration of the test may be increased in order to investigate longer

- term effects on the survival and reproductive success of the F 0 and/or F 1 and subsequent generations

.

6. When the copepods reach adulthood, which normally takes 15

- 18 days for the present species at
25C , the gender of each copepod is determined by light microscopy. Individual adult male:female mating pairs are then constructed and allowed to mate for 7

- 14 days in new isolated microw ell test chambers containing seawater and the test substance added to seawater at the same range of concentrations as during maturation/development. The mating pairs are followed until the females have been fertilised and have

released at least two clutche s of offspring. The reproductive output of the animals exposed to the test substance is compared to that of the control(s) in order to determine the reproductive no observed effect concentration (NOEC). In addition, and as far as possible, the data may be analysed using a regression model in order to estimate the concentration that would cause an x% reduction in reproductive output, i.e.,

EC

x ( e.g. , EC 50 , EC 20 or EC 10 )

. The mean development rates to the copepodite and adult stages, respectively

, survival of the parent animals

, and time to production of first and second clutch should also be reported. Other substance

- related effects on reproduction such as failed fertilization, depressed clutch sizes, infertile or unhatched eggs, altered time intervals betwee n successive clutches and possibly depressed intrinsic or instantaneous rates of population increase ( r m or λ ), may also be recorded or

calculated

.

DATA ON THE TEST SUBSTANCE

7. The water solubility, the vapour pressure, pK

a , and P

ow of the test substance should be known, and a reliable analytical method for the quantification of the substance in the test solutions with known and reported accuracy and limit of quantification should be available. Useful information for establishing the optimal test conditions includes the test substance structural formula, purity of the substance, stability in light, stability under the conditions of the test (abiotic and biotic degradation) and dissipation/fugacity. Loss of test compound (volatilizatio n, adsorption, accumulation, degradation, etc.) during the exposure period should be considered before starting the test. Further guidance for testing substances with properties that make them difficult to test with the described methods is provided in (12 ). Also personal safety precautions when working with the test substance should be taken. Waste containing the test substance should be treated in a way that does not cause human harm or environmental problems.

8. Results of an acute toxicity test (see ISO 1999 [ Water quality

- determination of acute lethal toxicity to marine copepods ( Copepoda

, Crustacea ). ISO 14669:1999. ]), preferably performed with the same species ( Amphiascus tenuiremis ), must be available. The results will be needed for selecting an appropriate range

of test concentrations in the definitive chronic tests ( See paragraphs 29 and 30 )

.

REFERENCE SUBSTANCES

9. Any of the reference substances CdCl 2 , lindane

, fipronil or fipronil sulfide may be t ested periodically as a means of assuring that the test protocol and test conditions are reliable, and that the sensitivity of copepod test stocks is consistent.

### 3 | Page VALIDITY OF TEST

10. For a test to be valid, the following performance criteria should be met :

 the average mortality in the control(s) must not exceed 2 5 % at the end of the exposure period; no individual control replicate microplates should exceed 30 % mortality.

 the dissolved oxygen concentration must have been at least 70% of the air saturation value (ASV) throughout the exposure period

 temperature should vary by no more than ± 4 °C during the entire test with a 36 day mean temperature of + 2.0 °C

 pH must not vary more than 0. 5 units from the average control pH with a 36 day mean pH of 8.0 +

0. 4

.

 c onductivity/salinity must not vary more than 10% from the control start value ( e.g. , 30‰ ± 3 ‰ )

 sex ratio in F 0 - controls (♀♀ : ♂♂) should on average range between 0.6 to 1.8 ( + 0.2)

.

 the average number of viable offspring produced through two clutch es in the controls should not

be less than 8 individuals

.

 the average number of days to the first clutch in the control(s) should at a temperature of 25°C not exceed the number of days representing 70% of the total exposure period or 25 days

, whichever is

less.

DESCRIPTION OF THE METHOD

Apparatus

11. Microwell test chambers and other test vessels and apparatus which will come into contact with the test solutions should, if possible, be made entirely of glass or other materials chemically inert to the test chemical. Microwell plates are typically made o f polystyrene plastic. Polystyrene is not well suited to testing with hydrophobic organic chemicals because of non

- specific plastic:organic binding, which can reduce chemical bioavailability to copepods. Therefore, microplates with microwells pre

- coated wi th glass or polyacrylamide gel are better suited for copepod testing of hydrophobic chemicals than microplates with naked polystyrene wells.

In addition, some or all of the following equipment will be required:

 oxygen meter (with microelectrode or other s uitable equipment for measuring dissolved oxygen in low volume ( e.g. , 10

- mL) samples);

 pH meter;

 conductivity meter, osmometer or salinity refractometer;

 adequate apparatus for the control of the lighting regime;

 stereo compound microscope, preferably inve rted with dark

- field or Hoffman DIC illumination;

 dissection stereomicroscope, preferably with dark

- field illumination and fiber optic episcopic

illumination.

### 4 | Page  climate

- controlled environmental chambers or a temperature regulated room;

 0.5 m 3 glass or plasti c rectangular containers with sealable lids to create humidity chambers for the microplates (to minimize evaporative loss).

 various plastic or stainless

- steel mesh sieves (0.05 mm to 0. 2 5 mm) to culture, capture and sort copepods;

 Pasteur pipettes, prefera bly silanized to prevent copepods from adhering to pipette walls;

 adjustable single and multi

- channel volumetric pipettors (1

- 10 μ L, 100

- 500 μ L) for food additions;

 250 μ L Hamilton glass volumetric syringes with 23 gauge beveled stainless

- steel needles;

 h a emacytometer cell counting chamber or electronic particle counter/analyser ( e.g. , a Coulter principle impedance

- based system)

Test organism

12. The species to be used in the test is Amphiascus tenuiremis

, which has widespread amphi

- Atlantic

distribution and occurs commonly in harpacticoid copepod field surveys. However, the taxonomic identification of this species may be difficult and therefore great care should be taken to correctly identify species used to establish laboratory cultures if collected in the f ield. It is recommended that test animals be obtained from already established laboratory cultures.

13. Test animals should be derived from a healthy stock ( i.e. , showing no signs of stress such as high mortality, poor fecundity etc). The stock animals must be maintained in culture conditions (light, temperature, seawater medium and feeding regimen) similar to those to be used in the test (a detailed culture method is contained in Annex 2).

Test medium

14. Natural or synthetic seawater may be used as th e test medium. If natural seawater is used, it should be collected from a location distant from any known sources of pollution and filtered to remove indigenous organisms. If synthetic seawater is used, it should be prepared by dissolving reagents of

recog ni z ed analytical grade, or a commercially available formulation, in distilled or deioni z ed water. However, for this species, there is insufficient information on the use of multiple synthetic seawater formulations to allow a particular brand or type to be recommended. The suitability of synthetic seawater for culture and testing should, therefore, be evaluated in trials. Any water in which the copepods show suitable long

- term survival with normal development and fecundity may be used as the test medium.

15. A. tenuiremis can be used at salinities between 18

- 40‰, but is normally cultured at a salinity of

20

- 30‰. The use of a lower salinity, which may be more appropriate for tests concerning estuarine brackish water situations, shall be justified in the test r eport. Whichever salinity is employed, the test organisms must be cultured or maintained at the same salinity (±3‰) for at least 7 days before the start of the test. The dilution water must have a dissolved oxygen concentration above 70% of the air saturat ion value, and a pH of 8.0 ± 0.3 before being used to prepare the test solutions. It is recommended that all

dilution water be filtered at 0.2 μ m immediately prior to test solution preparation to minimize bacterial

contamination.

### 5 | Page 16. The dilution water use d for testing should be from the same source as water that has been found to support culture of the organisms for at least two generations. If, however, the medium to be used in the test is different from that used for routine culture, it is a good practic e to include an adequate pre

- test acclimation period ( i.e. , sufficient time to culture the copepods through at least two generations) to avoid stressing the animals.

17. The dilution water should be of constant quality and samples should, if possible

, be taken at regular intervals for analysis. Measurement of heavy metals ( e.g. , Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations ( e.g. , Ca, Mg, Na, K, Cl, SO 4 ), pesticides ( e.g. , total organophosphorus and total organochlorine pesticides), total organic c arbon and suspended solids should be made, for example, every three months where a dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations can be less freque nt and intervals extended ( e.g. , every six months).

Test solutions

18. The test solutions of the chosen test chemical concentrations are usually prepared by dilution of a concentrated stock chemical solution. Stock solutions preferably should be prepared by dissolving the test

chemical in test medium. For substances with low water solubility, separate stock solutions should be prepared for the test chemical concentrations. The preferred options for preparing test solutions are physical methods, such as st irring and sonication (13). Saturation columns (solubility columns) can be used for achieving a suitable concentrated stock solution of poorly water

- soluble test chemicals.

19. The use of organic solvents may be required in some cases in order to produce a suitably concentrated stock solution, but every effort should be made to avoid the use of such materials. If solvents are used to produce a stock solution that can be dosed accurately into water, then the recommended

solvent concentration in the final tes t medium should not be greater than 1 mL/L, and it preferably should be the same in all test vessels. Use, where necessary, of solvents of low toxicity at low concentrations is recommended to aid preparation of media (13). The solvents listed below normally will not be toxic and

will not increase t he water solubility of a substance. Solvents may be essential in handling some substances; for example, for preparing stock solutions of hydrolytically unstable or highly viscous substances. Examples of suitable solvents are acetone, ethanol, methanol, dim ethylformamide, dimethylsulfoxide (DMSO), and triethylene glycol. Care should be taken when using readily biodegradable compounds ( e.g ., DMSO, acetone) as these can cause problems with bacterial build

- up and possibly lower dissolved oxygen in the test vess els.

20. The frequency of medium renewal will depend on the stability of the test substance. There must be evidence that the concentration of the test substance being tested has been satisfactorily maintained (see paragraph: “Frequency of analytical ...”). Test solutions should be renewed at 72

- h intervals or less.

PROCEDURE

Culture of copepods

21. The culture procedure for A. tenuiremis is described in Annex 2.

### 6 | Page Conditions of exposure

Duration

22. The test duration for the complete life

- cycle method is approximately 25

- 36 days (depending mainly on temperature and any developmental

- rate changes induced by the test chemical of interest), which is sufficient time for nauplii to develop to the adult stage, be mate d , extrude two clutches of eggs and produce offspring (the F 1 generation). Trials are usually ended after extrusion (and counting) of the second clutch of eggs. Hatching success of the first and second clutch of eggs should be determined, and the bioassay is usually terminated upon hatch of the se cond clutch, or on day 36, whichever occurs sooner

. Longer

- term observation of F 1 naupliar survival and development to adult will extend the total bioassay time by an additional 15

- 18 days. Extended observation is not required under this test guideline.

Loading

23. Copepods are, because of their small size, generally exposed in small volumes ( e.g. , <50 ml total ) of test solution. Still, larger volumes are usually prepared in order to meet minimum mass:volume requirements for physical

- chemical analysis ( e .g. , pH, DO, and determination of the test substance concentration). For this microplate based test, 100

- mL of test solution per concentration per water renewal ( see 32 below) should provide adequate volume for microplate

- based bioassay and chemical

anal ysis of most test substances.

Test animals

24. Newly hatched nauplii ( i.e. , within 24 hours) are obtained by isolating ovigerous females from a

mass culture at least 3 weeks old. At most

, 40 individually isolated test nauplii are added to separate micro wells dispersed 40 each among three or more replicate test vessels (microplates) per concentration and control(s). This will yield a maximum total initial test population per treatment or control of 120

individuals dispersed over triplicate microplates . It is not recommended that more than 40 individuals per microplate be assayed so that end and edge microwells can be avoided and left empty

. If a test population of > 120 individuals per t reatment or control is desired, then a fourth microplate should be added.

25. Treatments should be dispersed randomly among separate replicate microplates, as should all subsequent handling, loading, observation and feeding. Failure to do this may result i n bias that could be construed as being a chemical

- dependent effect . In particular, if experimental units are handled in treatment or concentration order, then some time

- related effect, such as operator fatigue or other error, could lead to greater or less er effects at the higher or lower concentrations. Only one test chemical concentration should be tested per microplate to reduce chances for microwell cross contamination. Furthermore, if the test results are likely to be affected by an initial or environm ental condition of the test, such as position in the laboratory or environmental chamber, then consideration should be given to blocking the test, or using a Latin

- square or similar experimental design.

Feeding

26. Copepod f eeding should be done at ev ery other microwell water renewal, i.e., every 6 th day of the

~ 36 day test

. Deviations from this schedule should be reported. When using small volumes of test

solution in semi

- static tests, it is important to consider the volume of food fed (particularly for weekly or more frequent feeding), and the potential for diluting the exposure concentrations. Food quality and quantity are critically important for optimal development and reproduction of copepods. Individual copepods in test chambers during maturati on/development and reproduction are fed 2 μ l of a 1:1:1 volume

mixed

- algal cell suspension (centrifuged, pelletized and re

- constituted in fresh 30 ‰ seawater to 10 7

### 7 | Page cells/mL) of a chlorophyte ( e.g. , Dunaliella tertiolecta ), a chrysophyte ( e.g. , Isochrysis galbana ) and a cryptophyte (e.g., Rhodomonas salina ) every six days during both maturation/development and reproductive periods. Therefore the cell density added to each test microwell at each feeding should target

~ 2 x 10 4 cells/microwell. This density will support copepod maturation and reproduction without fouling

the microwell environment. Specific details of the stock culture feeding regimes used for A. tenuiremis

are contained in Annex 2.

Light and temperature

27. During t esting, microplates are covered and placed in a temperature

- regulated incubator or room set at a desired controlled temperature (normally 25 ± 2 oC), with a 12

- h light and 12

- h dark photoperiod.

Aeration

28. The test vessels must not be aerated during the test, but dilution seawater should be aerated for 24

h , then filtered at 0.2 μ m
, and then mixed with test chemical stocks at the appropriate volumes and

concentrations

.

Test concentrations

29. Prior knowledge of the toxicity of the test substance ( e.g. , from an acute test and/or from range

finding studies) should help in selecting appropriate test concentrations. As a rule of thumb, the highest

concentration in the chronic test should be set between one tenth to one fifth of the LC 50 to avoid significant negative effects on survi val. This bioassay is a full lifecycle test so care must be taken to determine an appropriate dose range that will elicit effects of interest without reducing the test population to a number insufficient to provide at least ten or more mating pairs. Conc entrations that produce more

than 35% mor tality over the lifecycle must be reduced and re

- tested. Normally, there should be at least five test concentrations, each comprised of 20

- 40 separate micro wells dispersed among 3 replicate microplates per test su bstance concentration. Test concentrations should be arranged in arithmetic series with each successively lower concentration being 60% of the preceding higher concentration. For some test substances a geometric series with a concentration separation fac tor of 3.2 or less may be more appropriate depending on the slope of the dose response curve and the goals of the study. Justification should be provided if fewer than five concentrations are used. Substances should never be tested near or

above their s olubility limits in the seawater test medium.

When planning the test it should be taken into consideration whether the goal is to achieve a NOEC/LOEC (by use of ANOVA) or an EC

x values (by use of regression techniques). With an ANOVA approach, higher repl ication at the expense of fewer test concentrations often yields a more statistically robust experimental outcome. With regression techniques, more test concentrations at a finer scale of separation at the expense of fewer microplate replicates will often yield a more robust prediction of an EC

x .

30. In setting the range of concentrations, the following should be borne in mind:

(i) If the aim is to obtain the NOEC/LOEC, the lowest test concentration must be low enough so that the biological end point ( e.g. , fecundity) of interest at that concentration is not significantly lower than that of the control. If this is not the case, the test will have to be repeated with a reduced lowest concentration.

### 8 | Page (ii) If the aim is to obtain the NOEC/LOEC, the highest test concen tration must be high enough to cause a statistically significant effect when compared to the control for the biological end point ( e.g. , fecundity) of interest. If this is not the case, the test will have to be repeated with an increased highest concentrat ion.

(iii) If an EC

x for effects on reproduction is estimated, it is advisable that sufficient

concentrations be used to define the EC

x with the appropriate level of confidence. If the

EC 50 for effects on reproduction is estimated, it is recommended that the highest test concentration be greater than this EC 50 . Otherwise, although it will still be possible to

estimate an EC 50 , the confidence interval for the EC 50 will be unacceptably wide an d it may not be possible to satisfactorily assess the adequacy of the fitted model.

(iv) The range of test concentrations should preferably not include any concentrations that have a statistically and biologically significant effect on survival since the main o bjective of the test is to measure sublethal effects ( e.g. , on reproduction). The occurrence of mortality significantly higher than the control(s) in the test may require more complex statistical analysis.

Controls

31. One test

- medium control series and, i f relevant, one carrier control series containing the solvent carrier used to promote test substance solubility in seawater should be run. When used, the solvent control concentration should be set at the highest solvent concentration used in the microwell test chambers containing the test substance. This is determined most often as the solvent concentration required to solubilize the highest test substance concentration in seawater. The number of replicates in the control

and carrier control should be at least the same as for any test substance concentration; typically 60

- 120 microwells spread uniformly over 3 microplates.

Test medium renewal

32. The frequency of medium renewal in each test microwell will depend on the stability of the test substance, bu t it should occur at least every third day of the test. If, from preliminary stability tests, the test substance concentration is not stable ( i.e. , outside the range 50

– 150% of nominal or falling below 50% of the measured initial concentration) over the maximum renewal period ( i.e. , 3 days), consideration should be given to more frequent medium renewal. Potential evaporation losses of media from the test chambers should be monitored daily and, if needed, be corrected by more frequent medium renewal.

Whe n using micro well test chambers (300 μ L total volume ; 250 μ L working volume ), which are normally held in temperature

- regulated incubators or rooms (see paragraph 27), care must be taken so that the evaporation losses do not exceed 10% on a daily basis. T o prevent such a scenario, microplates must be placed on a platform over a water

- or sponge bath inside a humidity chamber in the temperature regulated incubator or room.

33. When the medium is renewed, a fixed volume (preferably 90

- 95% of the total test medium volume) should be aspirated from each microwell chamber under dissection stereomicroscopic observation and cool fiber optic or tangential lighting using a Hamilton gas s yringe of suitable needle diameter ( e.g. , 23 gauge beveled) . Most test media can be removed this way without drawing out the copepod. Care should be taken that copepods are not aspirated into the syringe. If copepods are aspirated they may experience st ress or egg sac damage; thus such aspirated copepods should be disqualified from

the test.

### 9 | Page Observations

34. Copepod lifecycle endpoints are monitored daily in each microplate using a stereoscopic inverted compound microscope with Kohler or better quality illumination (e.g., Hoffman differential interference contrast illumination) . A 4X objective lens with 10X ocular lenses will allow 100% field of view coverage of a single micro well through the bottom of the microplate. For sex determinations and egg s ac
counts, where desired, a 10X objective may be more helpful. Phase

- contrast or Hoffman DIC illumination makes microplate scoring , sex determinations and egg counting especially efficient at these magnifications. Test observations include mean developme nt rates from nauplius to the copepodite and adult stages, respectively, fertilization success ( i.e.

, number of mating pairs able to produce two successive clutches), total mean viable offspring production per mating pair ( i.e. through two clutches average d over the number of mating pairs created and tested), time to production of first clutch, time interval between

successive clutches

, and stage

- specific mortalities. Observations may also be made for any abnormal behaviour (relative to the controls), incl uding uncoordinated swimming, loss of equilibrium, change in pigmentation and atypical quiescence. Sex ratio may be determined by observations of male and female secondary sexual characteristics (see Annex 2 for details on how to determine differences betw een males and females), which in harpacticoid copepods normally can be distinguished in the 5 th copepodite stage at

the earliest. Numbers of aborted egg sacs and necrotic/infertile eggs may also be counted if desired.

35. Regular observations of F 0 - generation nauplii and copepodites as well as counting of F 1 - generation hatched nauplii must be facilitated by the use of light microscopy. An inverted stereomicroscope designed for microplate scanning is most suitable for this purpose (4

- 20X magnificati on ; see 34 above ). General sorting of gravid females for initial naupliar production and harvesting is best conducted under an upright dissection stereomicroscope with darkfield illumination and fiber optic episcopic illumination.

36. The results of the observations made during the test should be recorded on data sheets, examples of which are provided in Annex 3 with instructions on their use.

Development and Mating of Adults

37. The number of days it takes for development of each individually isolated c opepod, from a first stage nauplius to a juvenile copepodite to a reproductively mature adult, is checked daily. On the first test solution renewal day, when the copepods can be sexed visually (after 15

- 18 days), mature virgin males

and females are remove d from their respective treatment or control microplates and paired haphazardly with an opposite sex individual ( i.e. , from the same microplate within the same treatment) in a new or

unused micro well filled with test medium for each treatment or control replicate. If survival is 100% in a treatment or control up to the point of mating, then the yield of mating pairs will be approximately 50% of the starting naupliar n

- size if sex ratios are balanced. If sex ratios are skewed, or mortality rates have

pro duced insufficient numbers of males and/or females to produce multiple mating pairs within a given microplate within a given treatment, then virgin adults may be mated across microplates within treatments. However, this must be noted in any report of resu lts, and an a priori assumption of no significant microplate to microplate variability within the given treatment(s) must be made by the investigator.

Once individual mating pairs are established for each treatment and control collection of adults, 90

- 95% of transfer seawater should be aspirated (see 33 above) and replaced with the appropriate control or test

substance concentration. This replacement of media after matings are in place helps to ensure that transfer seawater is diluted to negligible levels at the beginning of the mating time window.

### 10 | Page Offspring production

38. After pairing of male:female mating pairs, observations are made daily for the presence of females carrying their first and subsequent egg sacs. In controls, two fertile egg sacs per female (> 4 eggs /sac normally ) are usually extruded simultaneously from the abdomen in 1

- 2 days. Offspring (nauplii) then will usually take two days to hatch

, but if more time is required, renewal of test medium and food should

be continued as described a bove (see paragraphs 32 and 33 under “Test medium renewal” ). Similarly, in the case of uneven sex ratios, spare unmated males or females may be retained and monitored for survival as individuals to the end of the test exposure period with renewal of test media continued as described

above. Total egg number per mating pair can be counted by direct observation of all eggs in each egg sac

if desired . Numbers of successfully hatching nauplii ( i.e., total viable clutch, or fecundity) should be

recorded for the first and second successive clutches of eggs. Hatching number can be obtained by subtracting any non

- hatched or necrotic eggs from the total clutch size at the clutch

- one and clutch

- two time points. Alternatively, numbers of hatched nauplii crawling on the bottom of the microwell may be counted by direct microscopic observation. Once the second clutch is hatched, then the y are counted and

the bioassay is complete for a given mating pair . A ll such mating pairs should be recorded as a

“success ful mating ” in the datasheet

. Percent mating success should be calculated for each group of mating pairs in each microplate within each treatment and control as the ratio of successful mating pairs versus unsuccessful pairs (i.e., those pairs unable to produce 2 via ble clutches of eggs) . An average percent mating success and standard deviation should be calculated from these data for each treatment and

control. Investigators may choose to extend the bioassay with F 1 to F N offspring depending on the needs and goals of the study. The mean number of viable offspring produced through two broods should be > 8 offspring per mating pair in the controls.

39. At the end of the exposure period, all remaining copepods ( i.e. , includin g unfertilised females and males) are either discarded , frozen or fixed in glutaraldehyde (4%) for determination of sex, toxicant body burdens, clutch sizes, egg quality, etc.

Mortality

40. Survival of larvae (nauplii), juveniles (copepodites) and adults are checked daily during maturation/development/mating up to and including the last day of the test. Mortality of mating pairs should be checked and recorded daily.

Other endpoints

41. Othe r endpoints that can be measured or calculated include time from appearance of first egg sacs

to release of first and second clutch, number of necrotic and infertile eggs per clutch

, numbers of dead nauplii, and morphological abnormalities. Indicators of i ntersexuality may also be observed using light microscopy (see Annex 2 for further information on easily observed secondary sexual characteristics in the present species). Population growth rate ( e.g. , the intrinsic (or instantaneous) rate of natural increase
[ r

m or λ ]) and age:stage structure can also be calculated and predicted over multiple generations using the resulting lifetable survival, sex

- ratio and fecundity data (see Annex 4 for further information).

Analytical determination and measureme nts

Physical

- chemical parameters

### 11 | Page 42. During the test, dissolved oxygen, pH and temperature should be measured in the controls and all test concentrations each time test medium is renewed. As a minimum, these measurements should be made in the control(s) an d in the highest test concentration. Temperature should preferably be monitored continuously in at least one blank test microplate or in a 100

- mL Griffin beaker filled with deionized water and placed inside the microplate humidity chamber.

Concentration of the test substance

43. During the test, the concentrations of test substance are determined at regular intervals. The concentration of the test substance is expected to remain within ± 50 % of the nominal values ( i.e. , within the range 50

– 150%). It is t herefore recommended that, as a minimum, the highest and lowest test concentrations and positive controls (if used) be analysed when freshly prepared at the start of the test and immediately prior to each renewal ( i.e., analyses should be made on a sample from the same solution

– when freshly prepared at the start and at each renewal).

44. It is recommended that results be based on measured concentrations. If there is evidence that the concentration of the substance being tested has been satisfactorily main tained within ± 20 percent of the nominal or measured concentration throughout the test, then results can be based on nominal or measured

initial values.

45. For tests where the concentration of the test substance is not expected to remain within ± 50 % o f the nominal concentration after 72 h or less, it is necessary to analyse all control(s) and test concentrations when freshly prepared and at test medium renewal. Used microwell seawater can be collected and pooled to yield

1

- mL for chemical analysis from every 4

- 5 microwells within a given treatment. In the case of rapid test chemical breakdown, results should be expressed in terms of the time

- weighted mean (see guidance for calculation in Annex 4). Note that care should be taken when testing very lipoph ilic ( i.e. , log K

ow > 5) and

hence poorly water

- soluble substances in the present test system (12). Use of radiolabelled substances can give crucial information on chemical partitioning in the test system, which may facilitate the verification of nominal concentrations. However, if the major fractions ( i.e. , >75%) of the radiolabelled substances are found to be adsorbed to particulate material ( e.g. , the food items) and/or walls of the test chambers, an alternative test system or methodology should be used, where the effect levels are related to contaminant levels in, e.g
., food.

DATA AND REPORTING

Treatment of results

46. If desired, acute mortality data for selected time periods ( e.g. , ≤ 96 hours) can be analysed based on the methods of Stephan (14) to calculate median lethal (LC 50 ) concentrations of test materials.

47. Statistically significant effects on survival of adult females are calculated using a contingency table procedure, Fisher's Exact Test (15), which should be reported as a separate endpoint. Those concentrations where mortality is > 35% are not included in the statistical analysis of the reproduction

data.

48. Larval/juvenile development rates, stage

- specific mortality, percent mating success, and sex ratios

of the F 0 may all be analysed statistically for significant differences relative to controls. Reproductive endpoints may be analysed as single end

- of

- test measurements or relative to different time intervals during the exposure. The mean number of F 1 offspring produced from the fertilised females (F 0 ) in each replicate

### 12 | Page is calculated. Generally, the reproduction data is not adjusted for mortality but are presented on a per female or per mating

- pair basis. The mean number of offspring produced by each control and treatment mating

- pair is calculated. If an animal dies after producing young, the total n umber of offspring produced up to the time of death is used. On occasions, it may be necessary to make an adjustment for mortality where those animals that died during the exposure ( i.e. , before giving birth to any young) are not included

in the statistica l analysis . One

- way nested analysis of variance (ANOVA) is used to evaluate effects of the test substance on the reproduction (and other chronic endpoints) of the copepods , and a multiple comparison procedure ( e.g. , Dunnett's test [16] is used to define th e treatment level resulting in e.g., significantly different reproduction from the control ( p = 0.05). For ANOVA, individual microwell mating pairs are nested within their particular microplate, which is then replicated within a given treatment. A

nested m odel is ideal if data are balanced across microplates and treatments because microwells within a microplate are not strictly independent of one another.

49. Analysis of variance is a parametric procedure and is based on the assumptions that the

observation s are independent and normally distributed, and that the variance of the observations is homogeneous across all toxicant concentrations and the control. These assumptions should be checked prior to using ANOVA, to determine if they have been met. Non

- norma l distributed data may be subjected to normalizing transformations such as log transforms, arc

- sine square root transforms, etc., provided both treatment and control datasets are subjected to the same transformation. Tests for validating these assumptions are Shapiro

- Wilks Test for normality and Bartlett's Test for homogeneity of variance (17). If the data does not meet the assumptions for ANOVA, non

- parametric procedures such as Steel's Many

One

- Rank Test or Wilcoxon's Rank Sum Test (17) may be more approp riate.

50. If a statistically significant difference in survival, development rate, or reproduction is detected between the control and solvent control, only the solvent control is used as the basis for the calculation of results. If no significant differe nces exist between control and solvent control data, these may be pooled

for greater statistical power in comparisons of control response with test substance treatments.

Test report

51. The test report must include the following information:

− reference to this Guideline

.

Test substance:

− physical nature and relevant physicochemical properties;

− chemical identification data (name, structural formula, CAS number, etc.) including purity;

− analytical method for quantification of the test substance where appropria te;

− supplier or source of the test substance.

Test species:

− the species of copepod used, supplier or source (if known) and the stock culture conditions used.

### 13 | Page Test conditions:

− test procedure used ( e.g. , microplate based static renewal)

− photoperiod and light intensity;

− test design ( e.g. , test concentrations used, number of replicates, number of copepods per replicate, number of mating pairs constructed per treatment);

− method of preparation of stock solutions and frequency of renewal (the solubilising age nt and its concentration must be given for each treatment, when used);

− the nominal test concentrations, the means of the measured values and their standard deviations in the test vessels and the method by which these were attained, evidence that the measur ements reflect concentrations of the test substance in true solution;

− dilution water characteristics (including pH, salinity, temperature, dissolved oxygen concentration, total organic carbon, suspended solids, and any other measurements made);

− detailed in formation on feeding ( e.g. , type of food(s), source, amount given, frequency of feeding,

and analyses for relevant unintended contaminants ( e.g. , PCBs, PAHs and organochlorine pesticides).

Results:

− results from any preliminary studies on the chemical stability of the test substance;

− the nominal test concentrations and the results of all analyses to determine the concentration of the test substance in the test vessels; the recovery efficiency of the analytical method and the

limit of detection should al so be reported;

− water quality of test media introduced to test vessels ( i.e. , pH, temperature and dissolved oxygen concentration;

− a full record of all the biological effects which were observed or measured and the statistical techniques used to analyse the data;

− the Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) for the biological end point(s) employed;

− ECx, where appropriate, and statistical methods used for its determination;

− other observed effects;

− an explanation for any and all deviations from the Test Guideline.

LITERATURE

(1) OECD 1995. Organisation for Economic Cooperation and Development. Aquatic Testing Methods for Pesticides and Industrial Chemicals . OECD.

### 14 | Page (2) OECD 1998. Organisation for Economic Cooperation and Development. Detailed review paper on aquatic testing methods for pesticides and industrial chemicals (Part 1: Report) , OECD series on testing and assessment (No. 11).

(3) Hicks, G.R.F., Coull, B.C. 1983. The eco logy of marine meiobenthic harpacticoid copepods. Oceanography and Marine Biology Annual Review 67

- 125.

(4) McKinnon, A.D., Duggan, S., Nichols, P.D., Rimmer, M.A., Semmens, G., Robino, B. 2003 The potential of tropical paracalanid copepods as live feeds in aquaculture. Aquaculture 223 (1

- 4), 89

- 106.

(5) Breitholtz, M., Bengtsson, B

- E. 2001. Oestrogens have no hormonal effect on the development and reproduction of the harpacticoid copepod Nitocra spinipes

. Marine Pollution Bulletin 42(10),

879

- 886.

(6) Breitholtz, M., Wollenberger, L., Dinan, L. 2003. Effects of four synthetic musks on the life

- cycle of the harpacticoid copepod Nitocra spinipes

. Aquatic Toxicology. 63(2), 103

– 118.

(7) Hutchinson, T.H., Pounds, N.A., Hampel, M., Williams, T.D. 1999. Impact of natural and synthetic steroids on the survival, development and reproduction of marine copepods ( Tisbe battagliai ). The Science of the Total Environment 233 , 167

- 179.

(8) Hutchinson, T.H., Pounds, N.A., Hampel, M., Williams, T.D. 1999. Life

- cycle stud ies with marine copepods ( Tisbe battagliai ) exposed to 20

- hydroxyecdysone and diethylstilbestrol. Environmental Toxicology and Chemistry 18 (12), 2914

- 2920.

(9) Chandler, G.T., Green, A.S. 2001. Developmental stage

- specific life

- cycle bioassay for

assessme nt of sediment

- associated toxicant effects on benthic copepod production. Environmental Toxicology and Chemistry 20, 171

- 178.

(10) Chandler, G.T., Cary, T.L., Volz, D.C. , Walse, S.S., Ferry, J.L., Klosterhaus, S.L. 2004. Fipronil effects on copepod development, fertility, and reproduction: A rapid life

- cycle assay in 96

- well microplate format. Environmental Toxicology and Chemistry 24, 177

- 124.

(11) ASTM E 2317

- 04. 2004

. Standard guide for conducting renewal microplate

- based life

- cycle toxicity tests with a marine meiobenthic copepod. American Society for Testing and Materials, West Conshohocken, PA, 22 pp.

(12) OECD 2000. Organisation for Economic Cooperation and De velopment. Guidance document on aquatic toxicity testing of difficult substances and mixtures. OECD series on testing and assessment (No. 23), Paris, France, 53 pp.

(13) Rufli, H., Fisk, P.R., Girling, A.E., King, J.M.H., Länge, R., Lejeune, X., Stelter, N., Stevens, C., Suteau, P., Tapp, J., Thus, J., Versteeg, D.J., Niessen, H.J. 1998. Aquatic toxicity testing of sparingly soluble, volatile, and unstable substances and i nterpretation and use of data. Ecotoxicology and Environmental Safety 39, 72

- 77.

(14) Stephan C.E. 1977. Methods for calculating an LC 50 . Proceedings 1st annual symposium on aquatic toxicology. Aquatic Toxicology and Hazard Evaluation. Ed: Mayer F L, Hame link J L, ASTM, 1977, STP 634, 65

- 84.

### 15 | Page (15) Finney D.J., Latscha, R., Bennett, B.M., Hsu, P. 1963. Tables for testing significance in a 2 × 2 contingency table. Cambridge University Press, London.

(16) Dunnett, C.W. 1955. Journal of American Statist i c al Association 50, 1096.

(17) USEPA 1988. Short

- term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. US Environmental Protection Agency, Office of Research and Development. EPA

- 600/4

- 87/028.

### 16 | Page 

## ANNEX 1

Definitions

Nauplii = Larvae

Copepodites = Juveniles

LOEC The lowest observed effect concentration is the lowest concentration within the experimental range at which a significant effect is observed.

NOEC

The no observed effect concentration is the tested concentration just below the LOEC.

EC

Effective Concentration

ECx

EC

x is the calculated concentration from which an effect of x% is expected. ECx is a function of time.

Confidence intervals

A x% confidence interval is an interval of values within which the measured or calculated value is likely to be present with a probability of x%.

Offspring production

Number of viable offspring produced per time unit Reproductive output Number of eggs (or offspring) produced per time unit

### 17 | Page 

## ANNEX 2

Biology and cultivation of Amphiascus tenuiremis

The organism

A. tenuiremis c.f. Mielke is an easily

- cultured, diosaccid harpacticoid copepod that is amphi

- Atlantic in distribution ranging from the North Sea/Baltic intertidal to the southern Gulf of Mexico (1). It is an epifaunal to deep burrowing (0 to 15 mm) species that ingests sediments for diatoms and bacteria, and produces burrowing larvae with no swimming ability. Sexes are dimorphic with females reaching 0.4 mm in length and males 0.25 to 0.30 mm. Males are also streamlined in shape and have a swollen geniculate

segment on their first antenna to clasp the female. Figure 1 below is a composite micrograph of each of the major life stages of this copepod.

Figure 1. Photomicrographs of Amphiascus tenuiremis major life stages.

A. tenuiremis has a generation time (egg to egg) of 17

- 18 days at 25 ° C in microplate culture, or 14

- 15 days in 25 ° C sediment culture. At 21 ° C in sediments, the generation time is 20

- 21 days egg to egg. Females are sexually mature after the fifth copepodite stage (that is, in the twelfth life stage) . They then mate and produce their first clutch in two to three days. Sperm transfer is via a membranous spermatophore sac attached externally by the male to the female genital pore. Isolated virgin females can be fertilized successfully up to 14 days post adulthood, and females will not extrude viable eggs unless paired with a reproductively

- functional mature male. Females will produce eight to nine clutches over their median lifetime of 49±2 days. Nauplii hatch within two to three days of egg extrusion a nd reach the copepodite stage in six to seven days.

Culturing

### 18 | Page General

Successful harpacticoid copepod culture is keyed by three important conditions: (a) a toxicant

- free muddy sediment substrate consisting of clay, silt, and fine sand particles less than 0.100 mm in size (median grain diameter approximately 0.004 to 0.02 mm); (b) a highly

- filtered carbon

- polished seawater source saturated with oxygen and relatively free of ammonia (less than 30 μg

- NH 4 /L; pH = 8.0 to 8.3); (c) a

varied diet of at least three easily cultured phytoplankton species (for example, a chlorophyte, Dunaliella

tertiolecta , a chrysophyte, Isochrysis galbana , and a diatom, Phaeodactylum tricornutum ). All marine algae are cultured in nutrient

- enriched seawater and can be purchased from the University of Texas

Culture Collection. Copepods can be purchased from Dr. G. T. Chandler at the University of South Carolina (tchandler@sc.edu). All copepods in a test should be from the same brood stock. Before a test is initiated, at least two previous generations should have been raised in sediments from birth using the same food, seawater, and approximate temperature as that to be used in the life

- cycle test. This will not only acclimate the copepods, but will also demonstrate the accepta bility of the food, seawater, and culturing procedures before the test is begun. Copepods may be grown continuously as static renewal sediment cultures provided the culture water is changed twice weekly (80% renewal) and continuously aerated.

Preparation of Clean Stock Sediments

Stock sediments for harpacticoid culture are prepared (after Chandler 1986 [2]) as follows: Collect

toxicant

- free muddy sediments from a pristine estuarine habitat (for example, North Inlet, SC, USA) and pass them through 0.5

- mm and 0.1

- mm sieves into deionized water (~1 kg sediment per 5L water). Let sediments settle for 1 hour, remove the supernatant, and then resuspend in fresh DI water. Repeat this wash at least three times after settling, and then centrifuge (3 min at 4000 R CF in 500

- mL centrifuge bottles) to remove 50 to 60 % of the water. Homogenize “dry” sediment pellets in a solvent

- cleaned

electric blender for 3

- 5 minutes, and then aliquot 600

- mL of the homogenate into 1

- L Griffin beakers, cover loosely with aluminum fo il, and autoclave for 15 min at 125C and 30 psi. Store autoclaved sediment stocks sealed at 4oC until use. Prepare culture sediment substrate by taking 100 g autoclaved stock, blending it for 3 min at high speed with 300

- mL 5

- μm

- filtered seawater, and then pouring the blended stock through a 0.063

- mm sieve into 3.5 L of filtered seawater in a 4

- L Griffin beaker. Allow sediments to settle for 30 min, and then aspirate away the supernatant water. Resuspend the remaining settled sediments again in 3.5 L filte red seawater and allow to resediment for 24 h at 4oC. After 24 h, aspirate away all supernatant water, leaving approximately 300 mL wet sediment slurry. The sediment to water ratio for copepod cultures should be approximately 1:100 for static renewal cultu res, or up to 1:20 for

flow

- through cultures. Wet sediment media has a consistent water

- to

- solids percentage of 87:13 to 88:12 using this technique. Sediment organic carbon content (based on C:H:N analysis) consistently ranges from 3.8 to 4.2 % among North Inlet, SC, USA sediment lots after passing them through these procedures. Raw North Inlet field sediments range from 4 to 6 % organic carbon by C:H:N. Final organic carbon content in wet sediment culture media will vary depending on the organic carbon co ntent of the raw source

sediments.

Inoculation, culture and feeding of copepods

Wet sediments (see above) should be poured slowly into 1

- L crystallizing dishes filled with 500 mL

seawater and allowed to settle for 1 h. Sediments are then inoculated with the copepod taxon of interest. Typically greater than 100 gravid copepods are required to start a viable culture. Flush each copepod culture dish slowly (approximately 3 to 5 mL/min) but continuously with polished natural or synthetic

seawater from a rec irculating (3) or ambient 0.005

- mm filtered seawater system, or replace 80% of the culture seawater twice weekly with fresh aerated seawater. Feed cultures a 1:1:1 mixed algal cell suspension in slight excess of what they consume (for example, approximate ly 2.5 · 10 8 centrifuged cells

### 19 | Page for a mature culture) twice per week. Culture algal cells to exponential growth using standard f/2 enriched

seawater media (4) and centrifuge for 12 min at 4700 RCF. Remove the supernatant, resuspend algae in

~1

- mL clean sed iment slurry (see above) and 50

- mL filtered seawater, microwave for 15 s (@ 800 W) to destroy swimming abilities, cool to 4oC, and pipette 10

- 50 mL into each culture depending on consumption rates. Cultures should not be fed more than they will visually co nsume in 24

- 48 hours. One hundred gravid copepods will often yield a peak

- density culture (>10

, 000 copepods) in 2 to 3 months if food, sediment, and water quality are high.

Harvesting stock cultures

A

. tenuiremis should be handled as little as possible. When handling is necessary, it should be done gently, carefully, and quickly so that the copepods are not unnecessarily stressed, and gravid females will not drop their externally carried egg sacks. When collectin g gravid A

. tenuiremis females, monoculture stock sediments should be sieved gently over a 125 μm stainless steel sieve, with the retained sediment fraction and copepods washed into a sterile, plastic petri dish (10 cm diameter) using seawater at the same salinity

as used for the life

- cycle test. High

- quality borosilicate glass Pasteur pipettes 146 mm in length should be

used for collection and transfer of the > 125 μm sieve fraction containing adult and gravid female A

. tenuiremis to 50

- mL glass crystalli zing dishes using a research

- grade dissection stereomicroscope and

cool fiber

- optic illumination. Copepods should be introduced into seawater solutions beneath the air to water interface. Copepods that touch dry surfaces or are dropped or injured should be discarded.

Harvesting Young for Testing

Less than 24

- h old young (nauplii) are obtained by first collecting adult and gravid females as stated above, and then apportioning ~ 100 gravid females equally among each well of a six

- well, polystyrene

NetWell plate filled individually with 15 mL of 0.2

- μm filtered seawater. Within each well, a 7 3

- 75

- μm nylon mesh basket (Corning Costar NetWells) is suspended to retain females approximately 3 mm above the well bottom. The basket allows newly hatched non

- swimming nauplii to fall through the mesh and away from adult females (see Fig. 2A below). At 24 hours, the mesh basket and post

- hatch females are removed, and nauplii are collected from all wells and transferred individually to 96

- well microplate microwells using silanized fine

- tipped (0.1 mm diameter) borosilicate glass P asteur pipets . After all wells are successfully loaded with a single nauplius, the overlying transfer seawater is removed under a stereomicroscope using an analytical grade 250

- μL glass syringe so that approximately 5

- 10 μL of seawater remains. This standardizes the starting volum e in each well and allows for minimal dilution of the test solutions. Once the overlying water is removed, 2 5 0 μL of control(s) or test solution is added, aspirated under microscopic viewing, and then added again to the appropriate wells using an appropria te volumetric micropipettor or microsyringe. After test solutions have been added, each well should receive 2 μL of a fresh, 10 7 cells·mL

- 1 1:1:1 mixed algal cell suspension as described previously.

Quality Assurance

To increase the chances of an acceptable test, the test should not begin with nauplii that were from injured or dying (that is, immobile) adult females . All nauplii should be handled and checked for viability using

cool light microscopy. All 24

- h old naupl ii must be alive before beginning the test. Nauplii are non

swimming so their survivability can be checked under a stereomicroscope by gently tapping on the p etri dish holding the representative nauplii and checking for appendage movement. Representative copepods from the brood stock should be analyzed for the test material of interest before beginning a test if there is a possibility that the test material might be present in the culturing environment.

### 20 | Page Figure 2. Schematic of (A) naupliar collection a pparatus, and (B) microplate test layout and design.



### Literature

(1.) Lang, K. 1948. Monographie der Harpacticiden . Hakan Ohlsson, Lund, Sweden.

(2.) Chandler, G.T. 1986. High density culture of meiobenthic harpacticoid copepods within a muddy

sedime nt substrate. Canadian Journal of Fisheries and Aquatic Sciences 43, 53

- 59.

(3.) Chandler, G.T., Green, A.S. 1996. A 14

- day harpacticoid copepod reproduction bioassay for laboratory and field contaminated muddy sediments, IN: Ostrander, G. (ed) New Techni ques in Aquatic Toxicology , CRC Press, Inc., Boca Raton, FL, 23

- 39.

(4.) Guillard, R.R.L. 1972. Culture of phytoplankton for feeding marine invertebrates. IN: Culture of

Marine Invertebrate Animals , Plenum Press, New York, NY, pp. 20

- 60.

### 21 

## ANNEX 3

Example data sheets for recording major endpoints (assuming the use of 96

- well microplates)

Daily log sheet tem plate (note: data are recorded as read in the microplate microwell row:column positions.)

Treatment:

1 Microplate I 1 2 3 4 5 6 7 8 9 10 11

A

B

C

D

E

F

G

H

2 Microplate II 1 2 3 4 5 6 7 8 9 10 11

A

B

C

D

E

F

G

H

3 Microplate III 1 2 3 4 5 6 7 8 9 10 11

A

B

C

D

E

F

G

H

### 22 Mating log sheet template

Test Material: Start

Date Start

Time Technician in Charge:

Concentration: Replicate: One

♀ ♂ Cell # Day

Mated Day of 1 st

Extrusion # Naups on 1 st

Extrusion 2 nd Day of

Extrusion # Naups on 2 nd

Extrusion Days b/t

Extrusions

1

2

3

4

5

6

7

8

9

10

11

12

13

Concentration: Replicate: Two

♀ ♂ Cell # Day

Mated Day of 1 st

Extrusion # Naups on 1 st

Extrusion 2 nd Day of

Extrusion # Naups on 2 nd

Extrusion Days b/t

Extrusions

1

2

3

4

5

6

7

8

9

10

11

12

13

Concentration: Replicate: Three

♀ ♂ Cell # Day

Mated Day of 1 st

Extrusion # Naups on 1 st

Extrusion 2 nd Day of

Extrusion # Naups on 2 nd

Extrusion Days b/t

Extrusions

1

2

3

4

5

6

7

8

9

10

11

12

13

### 23 

## ANNEX 4

Calculations

Population growth rate

There exist s numerous statistical techniques on how to calculate a population growth rate from individual

life

- table endpoints. The following references (1

- 5) and Figures (1

- 3) describe ho w to use Leslie matrices, which are powerful and relevant when studying individually isolated copepods over a full or multiple life cycle s :

(1) Leslie, P.H. 1945. On the use of matrices in certain population mathematics. Biometrika 33

, 183

- 212.

(2) Caswell, H. 2001. Matrix Population Models

– Construction, analysis, and interpretation (2 nd

edition) , Sunderlands, MA, Sinauer Associates.

(3) Ferson, S.; Ginzburg, L.; Silvers A. 1989. Extreme event risk analysis for age

- structured populations

. Ecol . Model

. 47 , 175

- 187.

(4) Power, M.; Power, G. 1995. A modelling framework for analyzing anthropogenic stresses on brook trout ( Salvelinus fontinalis ) populations. Ecol. Model

. 80 , 171

- 185.

(5) Akçakaya, H.R., Burgman, M.A., Ginzburg, L.R. Applied Population Ecology: Principles and Computer Exercises using RAMAS EcoLab 2.0 (2 nd Edition) . Setauket, NY:Applied

Biomathematics. 1999.

(6) Akçakaya, H.R., Stark, J.D., Bridges, T.S. 2008. Demographic Toxicity: Methods in ecological risk assessment. Oxfo rd Univ. Press, 288 pp.

### 24 6

- well plate with 75 μ m meshed

- well inserts;

Yields ~ 500 synchronous nauplii in < 24 hour

Amphiascus

nauplius

Life

- cycle Endpoints: Survival & Molting Success Development Rate

Time to first Copepodite

Time to Adult

Sex Ratio of F 0 Fertilization Success Clutch Size & Egg Quality

Abortion rates Offspring Production thru 2 Broods Hatching Success > 3 00 gravid A. tenuiremis

(from lab stock cultures)

(n > 60 nauplii/treatment) C 1



### X

- matings

Figure 1. Microplate

- based copepod lifecycle test for seawater

- borne reproductive and endoc rine toxicants. (after Chandler et al. 2004).

### 25 Figure 2. Example lifecycle graph of Amphiascus tenuiremis life

- stage development times showing the proportions of each life stage progressing (or failing to progress) to the next life stage through 24 days (i.e., the time required for controls to produce two hatche d broods of nauplii) at 25oC in control microplates. All proportions are exclusive of mortality (i.e., dead individuals were not scored as developmental failures). 

# mate fecundity hatch P ee 

## = 0.14

P cv 

## = 0.51

P vg 

## = 0.90



# Adult Virgin

Female

P
vv 

## = 0.10 F en 

## = 11.86 

# Gravid

Female Embryo P cc 

## = 0.01 P nn 

## = 0.09

P en 

## = 0.86 P nc 

## = 0.91 

# Nauplius Copepodite

2 days 8 days

7 days

1 day 

# C

O

N

T

R

O

L

R

E
S

P

O

N
S

E P
ee 

## = proportion of embryos not hatching

P
en 

## = proportion of embryos hatching to nauplii

P
nc 

## = proportion of nauplii becoming copepodites

P
nn 

## = proportion of nauplii not developing to copepodites

P cf 

## = proportion of copepodites becoming virgin females

P
cc 

## = proportion of copepodites not developing to adult

P fg 

## = proportion of mated females becoming gravid

P ff 

## = proportion of mated females remaining barren

F
en 

## = fecundity through two broods of nauplii 

##  Lifecycle = 18

- 19 days egg to egg

 Avg. Lifespan = 47 + 2 days

 Avg. Single Clutch = 6.2 + 3.6

eggs  8

- 10 Clutches/Lifetime

### 26 Figure 3. Matriarchal stage

- structured Leslie (Lefkovich) matrix population growth model for copepod development and reproduction in water

- borne chemicals. Model yields N generation projections per control and toxicant concentration which are then compared

.

P's = proportion from one life

- stage surviving and moving to next life stage, w/wout exposure, through the empirical microplate life

- cycle. Stages observed daily on inverted compound stereo

microscope. 

### P gg 

### P vg 

### 0 0 0 0 P

vv 

### P
cv 

### 0 0 0 0 P
cc 

### P
nc 

### 0 0 0 0 P
nn 

### P
en 

### F gg 

### 0 0 0 P

ee



### N g 

### (t) N
v 

### (t) N
c 

### (t) N n 

### (t) N
e 

### (t)

X

N g (t+1) (gravids)

N

v (t+1) (virgins) N

c (t+1) (copedites) N

n (t+1) (nauplii) N

e (t+1)
(embryos 

### )



# =

Copepo

dite Nauplius Embryo

(Viable or

Non

- Viable)



### Adult

Gravids Adult Virgin

Females mate hatch 

### P

ee 

### P
cc 

### P
nn



### P
en



### P
cv 

### P
nc



### P vg



### P

vv 

### (failures) P gg 

### F ge 

### fecundity