EXP1313100

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 guidelines and under Good Laboratory Practices 

(GLPs) and are listed in Table 1.

Table 1. Ecotoxicological Studies with EXP1313100

Study Title Study 

Number

Test 

Guideline Result Attachment

Acute Toxicity Study in 

Daphnia magna with 

EXP1313100

505715 OECD 202 48-hour EL50 > 100 mg/L. 

NOELR: 100 mg/L, the highest loading 

rate tested

027

96-hour Acute Toxicity 

Study in Carp with 

EXP1313100

505716 OECD 203 96-hour LL50 >100 mg/L

NOELR: 100 mg/L, the highest loading 

rate tested (limit test)

028

Fresh Water Algal 

Growth Inhibition Test 

with EXP1313100

505714 OECD 201 72-hour EL50: > 100 mg/L (growth and 

yield)

72-hour NOELR: 100 mg/L , the 

highest loading rate tested

026

Daphnia magna, 

Reproduction Test with 

EXP1313100 (semistatic)

505720 OECD 211 NOELR: could not be established

029

Fish Early-life Stage 

Toxicity Test with 

EXP1313100 (semistatic)

505721 OECD 210 NOELR: 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 adding a weighed amount of EXP1313100 to 

1
the 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 study, 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 concluded 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, and 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 level 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, using 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 concentrations, 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 Daphnia 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 and 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 

nominal 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 effects 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 in 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 neonates. The 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 adequate 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 the 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 

2
that 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

It 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 (HPV) 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 Reporting. 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 

algae. 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 effects 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 Attachments 035 and 036. EPA’s conclusions for the Alkaryl 

Sulfonate Category are summarized in Table 2 below and are shown in PMN Attachment 034. 

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)

3 
Conclusions

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. 

4 
Study Summaries 

STUDY TITLE: Acute 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 measured in each test chamber at the 

beginning and end of the test and 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 concentrations 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 the 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 EXP1313100 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 carp (Cyprinus carpio) were 

exposed under static conditions to WAFs prepared at loading rates 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 limit 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 confirmation 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 0.39 g fish/L.

5
A 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. Samples 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 of 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 for 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 exposure 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 mortality or overt 

signs of toxicity was observed in any organisms, therefore the LL50 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 the green alga Pseudokirchneriella subcapitata. Six 

replicates of exponentially growing algal cultures per group were exposed to a WAF prepared at a 

loading 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 104 cells/mL 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 (M2) 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 each replicate of the respective test concentration. Subsequently, 1 mL of an algal 

suspension was added to each replicate providing a cell density of 104 cells/mL. 

The initial concentrations in the WAFs prepared at nominal loading rates of 1.0, 10 and 100 mg/L 

EXP1313100 were 0.37, 2.6 and 7.1 mg/L. The two highest loading rates 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 concentration 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. 

6
Test chambers were kept at 21-23°C and under TLD-lamps with continuous illumination of 90 μE.m-2.s-1. 

The pH was measured in each test solution at the beginning and end of the test. The controls contained 

test medium (M2) only. Analysis of the test substance 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 densities 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, was 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 to 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 EXP1313100 (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. There were 20 replicate test chambers for each test treatment and control group, with one

daphnid in each chamber. Daphnids were fed Chorella pyrenoidosa suspension daily. 

 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 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 the 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 control and high dose groups. Every work 

7 
day 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 recorded 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., <20% 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 the 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 intratreatment 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 eleven 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 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 concentrations to 59-78% of initial during the 48-hour periods prior to 

renewal was observed. However, EXP1313100 lacks any readily hydrolysable functional groups, 

therefore 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).

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 coincided 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 indicated 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 reproduction.

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 concentrations throughout the study (and in additional 

investigations presented in the study report). Therefore, no reliable NOELR could be established in the 

study. 

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

SUMMARY: The objective of this 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-day 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 (fathead 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 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 (adjusted ISO medium with a hardness of 180 mg 

CaCO3/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 and 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, temperature was 

monitored continuously. 

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 intratreatment 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 in the measured concentrations throughout the study 

period.

9 
A decrease in the measured test concentrations to 6-71% of initial during the 48-hour periods prior to 

renewal was observed. However, EXP1313100 lacks any readily hydrolysable functional groups, 

therefore 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 control). The dissolved oxygen concentration was generally >60% of the air saturation 

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. 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 concentrations, as evidenced by the 

variability in the measured concentrations throughout the study.

10
EXP1313100

Study Summaries – Environmental 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. Environmental Fate Studies with EXP1313100

Study Title Study 

Number

Test 

Guideline Result Attachment

An Activated Sludge 

Respiration Inhibition 

Test 

505719 OECD 209 EC50 > 1000 mg/L, the highest 

concentration tested. 032/033

Ready Biodegradability 

by the Carbon Dioxide 

Evolution Test Method 

505722 OECD 

301B

Final mean % Biodeg: 10 - 14

Readily Biodeg - No 031

STUDY TITLE: Activated Sludge Respiration Inhibition Test (Carbon and Ammonium Oxidation) 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 EXP1313100 on activated sludge 

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/rangefinding test included a control group, a reference group (3,5-dichlorophenol, a known 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. An 

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-Liter test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tapwater 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 activated sludge was obtained from a wastewater treatment plant that primarily treats domestic 

waste. The 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 

11
collection. Control vessels contained 16 mL of synthetic sewage, 250 mL 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 solution, 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 

minutes.

All criteria for acceptability were met and the study was considered valid. The respiration rates of the 

controls exceeded 20 mg O2/g activated sludge/hr. The coefficient of variation of the controls did not 

exceed 30%. The EC50s of the 3,5-dichlorophenol reference groups, 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 the 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 mg/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 test 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 EXP1313100. 

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 (CO2) produced from the biodegradation of 

the test substance. This value is expressed as a percentage of the theoretical amount of CO2 (TCO2) 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 biodegradation 

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 Sample 

module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were 

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

Two parallel test series were performed 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 

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mineral medium containing the microbial organisms and mineral components were mixed. Three CO2-

absorbers (bottles filled 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 CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and 

precipitated out as barium carbonate. The amount of CO2 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 measured 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 

CO2 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 continuously 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 14 days, the difference of duplicate values for 

percent degradation of the test substance were less than 20%, the total CO2 release in the blank did not 

exceed 40 mg/L and the total CO2 release in the inert carrier blank did not exceed 70 mg/L, and the 

inorganic carbon content of the test substance suspension in the mineral medium at the beginning of 

the test was less than 5% of the TC content.

The theoretical CO2 (ThCO2) production of EXP1313100 was 2.8 mg CO2/mg. The ThCO2 of sodium 

acetate was calculated to be 1.07 mg CO2/mg. In the toxicity control with direct addition to the test 

medium, more than 25% biodegradation occurred within 14 days (28%, based on ThCO2). Therefore, the 

test substance was assumed not to inhibit microbial activity. In the toxicity control with the test 

substance coated on granular silica gel, less than 25% biodegradation occurred within 14 days (13%, 

based on ThCO2), 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.

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