### EXP 1313100

Study Summaries

– Ecotox

The following is a summary of information on the aquatic toxicity of EXP1313100, which comes from laboratory studies with the test substance and read

- across to similar substances.

Laboratory Studies

Studies have been conducted on the acute toxicity of EXP1313100 on fish, daphnids, and algae. In addition, studies have been conducted on the chronic toxicity of the substance to fish and daphnids. These studies were conducted according to OECD test guid elines and under Good Laboratory Practices (GLPs) and are listed in Table 1.

Table 1. Ecotox icological Studies with EXP1313100

Study Title Study
Number Test Guideline Result Attachment

Acute T oxicity S tudy in Daphnia magna with

EXP1313100 505715 OECD 202 48

- hour EL50 > 100 mg/L.

NOEL R : 100 mg/L, the highest loading
rate tested 027

96

- hour A cute T oxicity
S tudy in C arp with

EXP1313100 505716 OECD 203 96

- hour LL50 >100 mg/L

NOEL R : 100 mg/L, the highest loading rate tested (limit test) 028

Fresh W ater A lgal

G rowth I nhibition T est with EXP1313100 505714 OECD 201 72

- hour EL50: > 100 mg/L (growth and yield)

72

- hour NOEL R : 100 mg/L , the highest loading rate tested 026

Daphnia magna

, R eproduction T est with EXP1313100 (semi

static) 505720 OECD 211 NOEL R : c ould not be established

029

Fish E arly

- life S tage
T oxicity T est with EXP1313100 (semi

static) 505721 OECD 210 NOEL R : 10 mg/L, the highest loading
rate tested 030

EL50: Loading rate resulting in 50% effects; LL50: Loading rate resulting in 50% lethality; NOELR: no observed effect loading rate

The test substance is a glassy solid with limited water solubility (0.02 mg/L determined by OECD 105; see report 505730 Attachment 010 ). Efforts at the laboratory to prepare test substance stock solutions in
various solvents (acetone, dimethylformamide, DMSO, and methanol) at concentrations sufficient for testing were unsuccessful. Therefore, water

- accommodated fractions (WAFs) were prepared for all of the aquatic toxicity tests. Test solutions were prepared by add ing a weighed amount of EXP1313100 to

1the appropriate medium or dilution water, stirring for 2 days, and filtering through 0.45 μm membrane filters. Analytical confirmation of the test solution concentrations was performed for all studies.

In the algal st udy, the nominal WAF loading rates were 1.0, 10, and 100 mg/L. The average measured concentrations were above the maximum solubility level at 0.27, 2.6, and 7.7 mg/L, respectively. No effects upon growth rate or yield were observed and it can be conclude d that EXP1313100 had no
effects at saturation.
In the acute toxicity study with carp, the nominal WAF loading rate was 100 mg/L and the average measured exposure concentration was calculated to be 0.0038 mg/L. No mortality or other effects were noted, a nd it can be concluded that EXP1313100 had no effects at saturation.

In the acute toxicity study with Daphnia magna , the nominal WAF loading rates were 1.0, 10 and 100 mg/L. The average measured exposure concentrations were above the maximum solubility le vel at 0.029, 0.60, and 0.21 mg/L, respectively. No immobility or other effects of exposure were noted, and it
can be concluded that EXP1313100 had no effects at saturation.

The chronic toxicity studies were conducted under static renewal conditions, usin g freshly

- prepared WAFs at intervals during the exposure period. Although it was the best option available, it was evident that the WAF preparation technique was not adequate for generating test solutions of EXP1313100 with consistent exposure concentrati ons, as evidenced by the variability in the measured concentrations throughout each study. In the fish early life

- stage study, the coefficients of variation (CV) for the mean measured concentrations were 67 and 103%, while they were 109 and 133% in the Da phnia magna chronic toxicity study.

In the fish early life

- stage study, fathead minnow eggs were exposed over 32 days to two WAFs at nominal loading rates of 1.0 and 10 mg/L, which were renewed every 48 hours. There were no statistically significant effects on embryonic or larval survival a nd on larval growth at the highest loading rate and therefore, the NOELR was 10 mg/L. It can be concluded that EXP1313100 had no effects at
saturation.

In the chronic toxicity test with Daphnia magna , neonates were exposed over 21 days to two WAFs at

nom inal loading rates of 1.0 and 10 mg/L, which were renewed every 48 hours. No significant mortality was observed in parental Daphnia

. Statistically significant effects on reproduction and growth were observed at loading rates of 1.0 and 10 mg/L. These ef fects appeared to be related to the adherence of the algae used as food to the outside of the organisms This likely reduced the ability of the daphnids to obtain sufficient food, which would i n turn account for the statistically significant reduction in growth and reproduction. Adherence of the algal cells occurred only during the first 2 weeks of exposure

, during which most offspring produced by the exposed parental Daphnia were immobile ; afterwards, the exposed Daphnia released predominately mobile neo nates. T he variability in the measured concentrations throughout the reproduction study precluded determination of a reliable NOELR. It was concluded that lower test concentrations could not be prepared, because the WAF preparation technique is not adequ ate for generating consistent concentrations of EXP1313100 at set loading rates

. In summary

, EXP1313100 is a glassy solid with limited water solubility. Aquatic toxicity tests were conducted using WAFs. The results indicate that no effects at saturation occur in acute toxicity tests with algae, Daphnia , and fish, with EL50 or LL50 values greater than the highest loading rate tested. Chronic toxicity tests, which included repeated analytical measurements, indicated t he difficulty of producing consistent WAFs with this test substance. No effects at saturation were seen in a chronic toxicity test with fish , with the NOELR at the highest loading rate tested (10 mg/L ; CV of 103% ) . Effects

2that were observed in a chronic test with Daphnia appear to be related to a physical phenomenon affecting the food; a reliable NOELR could not be established in this study due to high variability in the exposure concentrations (CV’s of 109 and 133%)

.

Read

- Across

I t is similar to the substances in the petroleum additive alkaryl sulfonate category, for which a set of robust summaries have been prepared under US EPA’s High Production Volume (H PV ) Challenge Program by the American Chemistry Council Petroleum Additives Panel (October 2005). In the HPV evaluation the Petroleum Additive Alkaryl Sulfonate category was defined as complex mixtures designated as Class 2 substances for TSCA Inventory Rep orting. Class 2 substances, in general, have a complex and variable composition depending on their source or methods of derivation. The sulfonated alkylbenzenes are manufactured by the sulfonation of either synthetic alkylbenzene substrates or naturally occurring alkylaromatic

- rich fractions of heavy lubricating oil base stocks derived from petroleum streams. The alkyl substituent group may vary in number (e.g., mono - or dialkyl), position (e.g., predominantly meta or para to the sulfonic acid position), chain length (e.g., C14 to C30) or in the degree of branching. Branched and linear alkyl groups of 20 or more carbons are used to enhance oil solubility. The substances in this category include sulfonic acids as well as their calcium, sodium, barium and magnesium salts.

This class 2 substance thus fits within the definition of the Petroleum Additive Alkaryl Sulfonate category and supports the use of this category’s testing data.

A number of the 12 substances in this category were evaluated for acute toxicity to fish, daphnids, and
al gae. The substances were tested using WAFs. No mortality was observed in any of the five fish studies (maximum loading rates of 100, 1000 or 10,000 mg/L). Five substances were tested (maximum loading rates of 100 or 1000 mg/L) with Daphnia , with minimal eff ects seen; overall the EL50 was >100 mg/L. Five of the substances were evaluated for algal growth inhibition, at maximum loading rates of 100, 1000 or 1500 mg/L. Overall the EL50 for algae was >100 mg/L. All results were based on nominal loading rates; analytical confirmation was not performed in any of these studies. The HPV submission
and robust studies are included as PMN Atta chments 035 and 036. EPA’s conclusions for the Alkaryl Sulfonate Category are summarized in Table 2 below and are shown in PMN Attachment 0 34.

Table 2. HPV Ecotoxicity Assessment Conclusions for Alkaryl Sulfonate Category
Title Result
Fish 96

- h LC50 No effects at saturation (water solubility limit) Aquatic Invertebrates 48

- h EC50 No effects at saturation (water solubility limit) Aquatic Plants 72

- h EC50 No effects at saturation (water solubility limit)

3Conclusions

Based upon laboratory data and read

- across information, it is concluded that EXP1313100 is practically

non

- toxic to aquatic organisms, with no effects at saturation.

Due to its physical properties (high Kow) it would be unlikely to partition out of the oil and into aqueous systems and thus is unlikely to pose a threat to aquatic organisms.

4Study Summaries

STUDY TITLE : Acu te Toxicity Study in Daphnia magna with EXP1313100 (Static)

SUMMARY : This study was conducted according to OECD Guideline 202 and under GLP. Daphnia

magna neonates, <24 hours old, were exposed under static conditions . A combined limit/range

- finding test was performed. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a WAF prepared at a loading rate of 100 mg/L and to a control. In addition, ten daphnids per group (two replicates, five daphnids per replicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L. The total test period was 48 hours and samples for analytical confirmation of exposure
concentrations were taken at the start and at the end of the test. The daphnids were not fed during the study.

All test solutions were prepared separately. Various loading rates of the test substance were added to
the test medium (M7) applying two days of magnetic stirring in order to reach maximum solubility. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter where after the clear and colorless WAFs were used for testing. Test chambers were kept at 18

- 22 °C and under a photoperiod of 16 hours light: 8 hours dark. Dissolved oxygen and pH were mea sured in each test chamber at the beginning and end of the test an d every 24 hours during the test. Organisms were observed for immobility at 24 and 48 hours after test initiation.

The samples were analyzed on the day of sampling. The initial concentrat ions in the WAFs prepared at loading rates of 1.0, 10 and 100 mg/L EXP1313100 were 0.029, 0.60 and 0.21 mg/L. These concentrations remained stable during the test period (88

- 98% of initial at the end of the test). The
concentrations obtained in the WAFs prepared at loading rates of 10 and 100 mg/L indicated that the maximum solubility in test medium was exceeded at these loading rates. This conclusion was based on the water solubility of 0.02 mg/L, which was determined in Project 505730.

The study met th e acceptability criteria prescribed by the protocol and was considered valid.

No effect on mobility was recorded at any of the loading rates of EXP1313100 tested.

The 48h

- EL50 was above a loading rate of 100 mg/L EXP1313100. The concentration of EXP131310 0 in the WAF prepared at a loading rate of 100 mg/L was determined to be 0.21 mg/L based on the initial exposure concentration. This concentration was considered to be above the maximum solubility of
EXP1313100 in the test medium.

STUDY TITLE : A 96

- hour acute toxicity study in carp with EXP1313100 (static)

SUMMARY : This study was conducted according to OECD Guideline 203 and under GLP. A range

- finding
test and a limit test were performed. For the range finding test, three c arp ( Cyprinus carpio ) were exposed under static conditions to WAF s prepared at loading rat e s of 1, 10 and 100 mg/L of EXP1313100 and a control for a period of 96 hours. No mortality or effects were observed, so the definitive test was
conducted as a limit test. For the limi t test, seven carp per concentration with a mean wet weight of 1.11 g and a mean total length of 3.3 cm were exposed to the WAF prepared at a loading rate of 100 mg/L and to a control. The total test period was 96 hours and samples for analytical confirma tion of exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test. Fish were not fed during the test. The total weight of fish per liter of water was 0.3 9 g fish/L.

5A loading rate of 100 mg/L was added to the test medium (adjusted ISO medium) applying two days of magnetic stirring in order to reach maximum solubility. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter where after the clear and colorless WAF was used for testing. The
controls contained adjusted ISO medium only. S amples for analysis were taken from the limit
concentration and the control at 0, 24 and 96 hours. Samples were stored in a freezer until analysis.

Test chambers were kept at 20

- 24 °C and under a photoperiod o f 16 hours light:8 hours dark. Dissolved

oxygen , temperature, and pH were measured in each test chamber at the beginning and end of the test and every 24 hours during the test. Aeration was introduced after one day of exposure. Organisms
were observed f or mortality and signs of toxicity at approximately 3.75

, 24 , 48, 72 and 96 hours after
test initiation.

In the limit study, no mortality or clinical effects were observed. Analysis of the samples taken at the start of the limit test showed a measured concentration of 0.0045 mg/L in the WAF prepared at a loading rate of 100 mg/L. This concentration slightly decreased to 72% of initial at the end of the test. The average exp osure concentration was calculated to be 0.0038 mg/L.

Test validity criteria were met (<10% mortality in controls and dissolved oxygen concentration maintained).

All organisms exposed to the test substance appeared normal , as did the controls

. No mortal ity or overt signs of toxicity was observed in any organisms, therefore the L L50 value is reported as >100 mg/L, the highest loading rate tested.

STUDY TITLE : Fresh Water Algal Growth Inhibition Test with EXP1313100.

SUMMARY : This study was conducted according to OECD Guideline 201 and under GLP. A combined limit/range

- finding test was performed with t he green alga Pseudokirchneriella subcapitata

. Six replicates of exponentially growing algal cultures per group were exposed to a WAF prepared at a
lo ading rate of 100 mg/L and to a control. In addition, three replicates per group were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L. The initial cell density was 10 4 cells/m L and the total test period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test. The test was performed under static
conditions.

Various loading rates of the test substance were added to the test medium (M 2 ) applying two days of magnetic stirring in order to reach maximum solubility. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter (Whatman) where after the WAFs were used for testing. WAFs prepared at loading rates of 1.0 and 10 mg/L were observed to be clear and colorless, while the WAF prepared at a loading rate of 100 mg/L was observed to be hazy. After preparation, volumes of 50 mL
were added to ea ch replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10 4 cells/mL.

The initial concentrations in the WAFs prepared at nominal loading rates of 1.0, 10 and 10 0 mg/L

EXP1313100 were 0.37, 2.6 and 7.1 mg/L. The two highest loading rate s remained stable during the test period (96

- 109% of initial at the end of the test), while the lowest loading rate decreased to 52% of
initial at the end of the test. The concent ration obtained in the WAF prepared at a loading rate of 100 mg/L was clearly above the maximum solubility in test medium based on the observed haziness of the
test solution. The average exposure concentrations were calculated to be 0.27, 2.6 and 7.7 mg/L

.

6Test chambers were kept at 21 -23 °C and under TLD

- lamps with continuous illumination of 90 μE.m -2 .s -1

. The pH w as measured in each test solution at the beginning and end of the test. The controls contained test medium (M2) only. Analysis of the test s ubstance concentrations and the control was performed on samples taken at 0, 24 and 72 hours. Samples were analyzed the day of sampling. At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densitie s were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank. In order to correct for turbidity one control per concentration, without algae, w as measured at each time interval.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

No inhibition of growth rate or inhibition of yield was recorded at any of the loading rates of
EXP1313100 tested.

The 72h

- EL50 for both growth rate and yield inhibition was above a loading rate of 100 mg/L EXP1313100 correspondingly the No Observed Effect Loading rate was 100 mg/L. The concentration of EXP1313100 in the WAF prepared at a loading rate of 100 mg/L was determined t o be 7.7 mg/L based on the average exposure concentration. This concentration was considered to be above the maximum solubility level of EXP1313100 in test medium.

STUDY TITLE: Daphnia magna

, reproduction test with EXP 1313100 (semi

- static)

SUMMARY : The purpose of the test was to evaluate the effects of the test substance on the mobility, the growth and the reproductive capacity of Daphnia magna

. This study was conducted according to
OECD Guideline 211 and under GLP. Daphnia magna neonates, < 24 hours old, were exposed under
semi- static conditions ( 48

- hour renewal) to loading rates of 1 and 10 mg/L of EXP1313100 for a period of 21 days. Every workday the condition of the parental daphnids was recorded, and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test

, the lengths of the surviving parental daphnids were
measured. T here were 20 replicate test chambers for each test treatment and control grou p, with one daphnid in each chamber. Daphnids were fed Chorella pyrenoidosa suspension daily

.

A non

- GL P investigation w as performed to identify a suitable solvent for preparation of a stock solution of EXP1313100 that would subsequently be used to prepare
the test solutions. EXP1313100 was not sufficiently soluble in any suitable solvent and was therefore
tested as WAFs

. Preparation of test solutions started with loading rates of 1.0 and 10 mg/L applying two days of magnetic stirring in order to reach maximum solubility. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter where after the clear and colorless WAFs were used for testing. The controls contained test medium only (M7). Samples for analytical confirmation of actual exposure concentrations were taken from both test concentrations and the control at the beginning and
end of three of th e renewal periods (days 0 and 2, 8 and 10, and 14 and 16)

. Samples were stored in the
freezer prior to analysis

. Test chambers were kept at 18

- 22 °C , constant to within 2°C, and under a photoperiod of 16 hours light:8
hours dark. Dissolved oxygen, temperature and pH were measured the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids. Water hardness
was tested once a week in fresh and old medium from the contro l and high dose groups. Every work

7day and upon renewal on non

- workdays, mortality and mobility of parental daphnids, the presence of eggs in the brood pouch, and the number and condition of neonates was recorded. Body length of parental daphnids was rec orded at the end of the test. The time to production of first brood and presence of unhatched eggs was recorded when observed.

Test validity criteria were met , i.e., <2 0% mortality in the control and the mean number of live offspring produced per parent animal surviving at the end of the test was >60

.
At t he start of each renewal period, the actual test concentrations ranged between 0.00037 and 0.0053 mg/L in the WAF prepared at 1.0 mg/L and between 0.00070 and 0.035 mg/L in the WAF prepared at 10 mg/L. This equates to a 14 fold intra

- treatment variability for the 1.0 mg/L WAF, and a 50 fold intra

treatment variability for the 10 mg/L WAF.

There are two main factors considered likely to have contributed to the observed variability in the test

concentra tions. First, the loading rates used were well in excess of the water solubility of EXP1313100 of 0.02 mg/L, which may have contributed to the presence of undissolved test substance in the WAF, which would have likely varied across the eleven renewal prep arations. and it was difficult to weigh amounts with a uniform surface area each time. T he surface area of the sample used to prepare the WAFs is likely to have affected the dissolution rates of
EXP1313100 durin g the preparation of each renewal media. Thus, even though the same weight of EXP1313100 was used to prepare each respective treatment at each renewal, different amounts of surface area would be expected to be present. This is likely to have contributed to variation seen in the measured concentrations throughout the study period. The mean measured concentrations were calculated to be 0.0019 and 0.011 mg/L, with coefficients of variation of 109% and 133%, respectively.

A decrease in the measured test conce ntration s to 59

- 78% of initial during the 48

- hour periods prior to
renewal was observed. However, EXP1313100 lacks any readily hydrolysable functional groups,
therefore th is decrease was not attributed to hydrolysis. The decrease may be due to precipitat ion and/or adsorption of the test substance (although no visible precipitation was seen).

There was no significant mortality of adults exposed to the test substance

. Statistically significant effects on reproduction and growth were observed at loading rates of 1.0 and 10 mg/L. Hence, a NOELR for both reproduction and growth of parental daphnids could not be established in this study. The effects on reproduction coinc ided with algal cells fed to the organisms adhering to the parental Daphnia

, a phenomenon noted from day 4 to 14 in both test treatments. After day 14 this phenomenon ceased and parental daphnids began to produce predominantly mobile offspring. This indi cated that the effects on reproduction were transient and related to parental daphnids being hampered by the sticking of the algal cells. The algae stuck to the outside of the organisms would not have been able to have been ingested and would have hampered the daphnids’ ability to access the remaining food in the test media, reducing the ability of the parental daphnids to obtain sufficient food. Reduced feeding ability would in turn account for the statistically significant reduction in growth and reprodu ction.

In conclusion, although it was the best option available, the WAF preparation technique was not adequate for generating test solutions of EXP1313100 with consistent exposure concentrations, as evidenced by the variability in the measured concentrati ons throughout the study (and in additional investigations presented in the study report). Therefore

, no reliable NOEL R could be established in the study.

8STUDY TITLE : Fish early life stage toxicity test with EXP1313100 (Semi

- static)

SUMMARY : The obje ctive of th is study was to define the lethal and sub lethal effects of the test substance on sensitive early life stages of the fish species tested. This study was conducted according to
OECD Guideline 210 and US EPA OPPTS 850.1400 and under GLP. A 32

- da y early life stage (ELS) test was performed under semi

- static conditions with renewal of test solutions every 48 hours. The test was performed with four replicates of a control group and four replicates for each WAF. Each test group contained 20 eggs (fa thead minnow ( Pimephales promelas )) per replicate. The test started by placing fertilized eggs in Petri -dishes. On days 6, 16 and 24 hatched larvae and/or juvenile fish were transferred to larger vessels/volumes. During the embryonic and larval phases, the fish were observed for effects on development, appearance and swimming behavior. At the end of the test (day 32), the surviving fish were measured and weighed. No feeding was provided during the embryonic phase. Newly hatched
larvae were fed Brachionus suspensions . Juvenile fish were fed brine shrimp Nauplii 24 or 48

- hours old. Food was supplied ad libitum.

The batch of EXP1313100 that was not completely soluble in test medium at the loading rates initially prepared. A non

- GLP investigation was performed to identify a suitable solvent for preparation of a stock solution of EXP1313100 that would subsequently be used to prepare the test solutions. EXP1313100 was not sufficiently soluble in any suitable solvent and was theref ore tested as WAFs. Preparation of test solutions started with loading rates of 1.0 and 10 mg/L applying two days of magnetic stirring in order to reach maximum solubility. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter whe re after the clear and colorless WAFs were used for testing. The controls contained test medium only ( a djusted ISO medium with a hardness of 180 mg

CaCO 3 /L and a pH of 7.7 ± 0.3 ). Samples for analytical confirmation of actual exposure concentrations

were taken from both test concentrations and the control at the beginning and end of five of the renewal periods (days 0 and 2, 6 and 8, 12 and 14, 20 a n d 22, 26 and 28 ). Samples were stored in the freezer prior to analysis.

The test temperature was 25± 1 .5 °C (with a few exceptions that did not affect study results) and the photoperiod was 16 hours light:8 hours dark. Dissolved oxygen, temperature, and pH were measured at
the start of the test and at each renewal in all test concentrations. In addition, t emp erature was monitored continuous ly

. At the start of each renewal period the actual test concentrations ranged between 0.0046 and 0.078 mg/L in the WAF prepared at 1.0 mg/L and between 0.017 and 0.46 mg/L in the WAF prepared at 10 mg/L. This equates to a 17 fold intra

- treatment variability for the 1.0 mg/L WAF, and a 27 fold intra

treatment variability for the 10 mg/L WAF. There are two main factors considered likely to have contributed to the observed variability in the test concentrations. First, the loading rates used were well in excess of the water solubility of EXP1313100 of 0.02 mg/L, which may have contributed to the presence of undissolved test substance in the WAF, which would have likely varied across the sixteen renewal preparations. and it was difficult to weigh amounts
with a uniform surface area each time. The surface area of the sample used to prepare the WAFs is likely to have affected the dissolution rates of EXP1313100 during the preparation of each renewal
media. Thus, even though the same weight of EXP1313100 was used to prepare each respective treatment at each renewal, different amounts of surface area would be expected to be present. This is likely to have contributed to variation seen i n the measured concentrations throughout the study period.

9A decrease in the measured test concentrations to 6

- 7 1 % of initial during the 48

- hour periods prior to
renewal was observed. However, EXP1313100 lacks any readily hydrolysable functional groups,

t herefore this decrease was not attributed to hydrolysis. The decrease may be due to precipitation and/or adsorption of the test substance (although no visible precipitation was seen). Based on these
results mean measured concentrations were calculated to be 0.015 and 0.14 mg/L for the whole test period of 32 days

. The coefficients of variation for these means were 67% and 103%, respectively.

The study generally met the acceptability criteria prescribed by the protocol and was considered valid

. Overall survival of fertilized eggs in the control was >70% until hatching was complete (i.e. 95% in the control). The overall survival of hatched larvae was >75% in the control during the remaining test period (i.e. 87% in the contro l). T he dissolved oxygen con centration was generally >60% of the air satur ation value throughout the test ; there was one exception which did not impact larval survival

. The water temperature generally did not differ by more than ± 1.5°C between test chambers between successive days at any time during the test, and was within the temperature ranges specified for the test species. Exposure concentrations during the test period were analytically monitored and study results were based on both loading rate and mathematical mean measured concentrations

. EXP1313100 did not induce any statistically significant effects on embryonic and larval survival and on larval growth at a loading rate of 10 mg/L. Hence, the NOEL and LOEL for this study, based on survival and growth rate, were 10 and > 10 mg/L, respectively. The concentration of EXP1313100 in the WAF prepared at a loading rate of 10 mg/L was determined to be 0.14 mg/L based on the mean exposure
concentration ; however, there was a large degree of variability in this concentration

. Altho ugh it was the best option available, it was evident that the WAF preparation technique was not adequate for generating test solutions of EXP1313100 with consistent exposure concentrations, as evidenced by the variability in the measured concentrations thr oughout the study.

10

### EXP 1313100

Study Summaries

– E nvironmental Fate

The following is a summary of information on the environmental fate of EXP1313100, which comes from laboratory studies with the test substance. These studies were conducted according to OECD test guidelines and under Good Laboratory Practices (GLPs) and are listed in Table 3

.

Table 3 . E nvironmental Fate Studies with EXP1313100

Study Title Study
Number Test Guideline Result Attachment

An A ctivated S ludge

R espiration I nhibition

T est 50571 9 OECD 20 9 EC50 > 1000 mg/L, the highest
concentration tested. 032/033

Ready B iodegradability by the C arbon D ioxide

E volution T est M ethod 5057 22 OECD
301B Final mean % Biodeg: 10

- 14 Readily Biodeg

- No 031

STUDY TITLE : A ctivated S ludge R espiration I nhibition T est ( C arbon and A mmonium O xidation ) with

EXP1313100

SUMMARY : This study was conducted according to OECD Guideline 209 and in compliance with GLP. The objective of the study was to determine the effect of EXP1 313100 on activated slu dge microorganisms by determining the respiration rate, and to calculate an EC50 and NOEC if possible. The study included a combined limit/range

- finding test followed by a limit test. The combined limit/range

finding test included a control group, a refe rence group (3,5

- dichlorophenol, a know n inhibitor of respiration, at 2 , 5 and 12 mg/L), and a treatment group, which included three concentrations of the test substance (10, 100 and 1000 mg/L nominal). The 1000 mg/L treatment was tested in triplicate. A n abiotic control (test substance at 1000 mg/L) was also included to discriminate between abiotic uptake by the test substance and microbial respiration . In the limit test a control group (6 replicates) , reference
substance (one replicate at each of three concentrations)

, and test substance concentrations (1000 mg/L) with and without a nitrification inhibitor (N

- allylthiourea) were tested in triplicate

. There was also a control group with the nitrification inhibitor (duplicates).

The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1

- Lit e r test bottles were filled with 200 ml of test substance mixtures in Milli

- RO water (tap

wat er purified by reverse osmosis ) with initial loading rates of 2.5 times the final loading rate. These
mixtures were stirred in closed dark brown bottles for 24 to 243⁄4 hours. Subsequently, 50 ml synthetic medium (= sewage feed), 250 ml sludge and Milli

- RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured by applying continuous aeration and stirring.

The a ctivated sludge was obtained from a wastewater treatment plant that primarily treats domestic

waste. T he total suspended solids were sieved, washed and diluted with ISO

- medium. The amount of suspended solids was determined to be 3,000 mg/L

. The batch of sludge was used one day after

11collection. Control vessels contained 16 mL of synt hetic sewage, 25 0 m L of sludge inoculum, and enough municipal water to bring the volume to 500 mL. Test substance or reference substance dosed biotic mixtures were prepared similarly but contained the appropriate amount of test substance or
reference substance stock solutio n, while the abiotic control was similar but contained no sludge. All mixtures were aerated for 3 hours (at 20 ± 2°C ; temperature during the limit test was below this but the controls met validity criteria ) after which the respiration rates were recorded for a period of 10

- 12
minute s.
All criteria for acceptability were met and the study was considered valid. The r espiration rates of the
controls exceeded 2 0 mg O 2 / g activated sludge /hr

. The coefficient of variation of the controls did not
exceed 30%

. T he EC50s of the 3,5

- dichlorophenol reference group s , were in the acceptable range (7.0 mg/L in combined limit/range finding test and 5.2 mg/L in the limit test).

In the combined limit/range

- finding test no inhibition of the respiration rate was observed at a loading rate of 10 mg/l, 6% was observed at 100 mg/L and an average of 1% was observed at 1000 mg/L. The respiration rate of one of the three replicates at th e highest loading rate could not be determined because of the absence of a linear part in the respiration curve. Therefore, an additional limit test was performed.
In the limit test no significant inhibition was observed at 1000 mg/L. Results at 1000 m g/L with a nitrification inhibitor showed that the heterotrophic respiration was 9% inhibited. Based on the results of the total and heterotrophic inhibition, nitrification was calculated not to be inhibited (

- 30%).

Under the conditions of this present te st EXP1313100 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.

STUDY TITLE : Determination of ‘Ready’ Biodegradability: Carbon Dioxide (CO2) Evolution Test (Modified Sturm Test) of EXP1 313100

.
SUMMARY : This study was conducted according to OECD Guideline 301B and in compliance with GLP. The objective was to measure the amount of carbon dioxide (CO 2 ) produced from the biodegradation of
the test substance. This value is expressed as a percentage of the theoretical amount of CO 2 (TCO 2 ) that could have been produced if complete biodegradation of the test substance occurred. Tests of ready biodegradability are stringent tests that provide limited opportunity for acclimation and biod egradation

to occur.

EXP1313100 was a brown solid and not sufficiently soluble to allow the preparation of an aqueous stock solution. Since the exact composition of the test substance was unknown at the start of the test, the organic carbon content could not be calculated. Therefore, a sample of EXP1313100 was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a
Shimadzu TOC

- VCPH total organic carbon analyzer combined with a Shimadzu SSM

- 5000A (Solid Samp le module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were

g lucose as total carbon (TC) standard and sodium carbonate as inorganic carbon (IC) standard.

The TOC content of EXP1313100 was determined to be 76%. The test substance was tested in duplicate at approximately 15.7 mg/L, corresponding to 12 mg TOC/L. The carbon content was based on TOC

analysis. No correction was made for the purity/composition of the test substance.

Two parallel test series were perform ed applying two different methods for the test substance addition. In the first series the test substance was added directly to the pre

- incubation medium and in the second

12mineral medium containing the microbial organisms and mineral components were mixed. Three CO 2 - absorbers (bottles fille d with 100 mL 0.0125 M Ba(OH) 2 ) were connected in series to the exit air line of each test bottle. Test medium without test substance or other additives (blank) and one control containing the granular silica gel used in preparation method to coat the test substance on granular silica gel (inert carrier

- blank) served as controls

.
The CO 2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO 2 produced was determined by titrating the remaining Ba(OH) 2 with 0.05 M standardized HCl. Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the blanks and test suspensions. Titrations for the positive and both toxicity controls were made over a period of at least 14 days. On day 28, the pH of all test suspensions was measure d and 1 mL of concentrated HCl was added to the bottles of both the blanks and test suspensions. The bottles were aerated overnight to drive off

CO 2 present in the test suspension. The final titration was made on day 29. The pH was recorded at the start of the test (Day 0) and on Day 28 before the addition of HCl

, and the temperature of the medium

was c ontinuously monitored in a separate vessel in the same room.

All criteria for acceptability were met and the study was considered valid. The positive control substance was biodegraded by at least 60% within 1 4 days, the difference of duplicate values for percent degradation of the test substance were less than 20%, the total CO 2 release in the blank did not exceed 40 mg/L and the total CO 2 release in the inert carrier blank did not exceed 70 mg/L, and the inorganic carbon content of the test substance susp ension in the mineral medium at the beginning of
the test was less than 5% of the TC content.

The theoretical CO 2 (Th CO 2 ) production of EXP1313100 was 2.8 mg CO 2 /mg. The Th CO 2 of sodium

acetate was calculated to be 1.07 mg CO 2 /mg. In the toxicity control with direct addition to the test medium, more than 25% biodegradation occurred within 14 days (28%, based on Th CO 2 ). Therefore, the test substance was assumed not to inhibit microbial activity. In the toxicity control with the test substance coated on g ranular silica gel, less than 25% biodegradation occurred w ithin 14 days (13%,
based on ThCO 2 ), and it was noted that the biodegradation levelled off after day 6. The biodegradation of the toxicity control with the test substance coated on granular silica gel was lower compared with the toxicity control with direct addition of the test substance to the test medium. However, inherent toxicity is not confirmed by the toxicity control with direct addition of the EXP1313100 and therefore it
be concluded that EXP1313100 is not toxic to micro

- organisms at the concentration tested.

Mean biodegradation at the end of the test with direct addition of EXP1313100 was 10%, while mean biodegradation at the end of the test with the test substance coated on silica gel was 14%. EXP1313100 was not readily biodegradable under the conditions of the modified Sturm test.

14