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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study according OECD 211 including analytical monitoring
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 h or 72 h old test solutions before renewal (combined sample from test vessels with daphnids).
Additionally, samples were collected from the 48 hour or 72 hour old abiotic replicate (no Daphnia or food algae) per test group.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is sparingly water soluble (≤0.019 mg/L). Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a saturated solution (water saturated fraction, WSF) of the test substance. The test solutions were weighed in and prepare separately (differential loading). Prior to use the test substance was melted at approx. 90°C. Then the test solutions were prepared by weighing test substance onto a plastic weighing boat and submerging it into 1L of test medium. The solution was stirred for approximately 96 hours to ensure that the saturation concentration is reached. Undissolved test substance was visible on the weighing boat.
Prior to toxicity testing, each solution was filtered (0.2 μm cellulose acetate membrane filter, Whatman OE 66) to assure the removal of any undissolved test substance. Filters were first wetted and rinsed with at least 100 mL of deionized water. Once wet, filters were not to dry out, so some residual water should be left standing over the filter. Approximately 800 mL of the test solution was then added to the filtration unit and the first 50 – 90 mL were discarded in order to saturate the filter. The remainder of the test solution was bulk filtered to the point where approximately 10 mL remains standing over the filter. The filtered test solution was used for toxicity testing. Control test media was similarly filtered.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978.
From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Age at test initiation: < 24 h
- Feeding during test: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic
medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1390 mg TOC/L and 1460 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤144 μL) of the concentrate to reach the amount of food defined below. The algae were stored in a refrigerator (dark, about 4- 8°C) for maximum 21 days. By adding the algal concentrate the test solution was slightly diluted. During a 72 h test interval with 3 feedings, 0.144 mL each, a total of 0.432 mL are added to 50 mL test volume resulting in a maximal dilution of 0.86%.
- Feeding schedule:
Day of Test Amount of food per parent animal and day [mg TOC] *
0-3 0.10
4-5 0.12
6-7 0.14
8-21 0.20
*amount of feed algae determined as total organic carbon.

ACCLIMATION
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are
used for testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
22 d
Hardness:
2.20 – 3.20 mmol/L
Test temperature:
20 ±1°C
pH:
7.5 – 8.5
Dissolved oxygen:
Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.
Nominal and measured concentrations:
1.2, 2.0, 3.5, 5.9, 10 mg(L (nominal; loading rates)
Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.
- Test volume: 50 mL
- Aeration: none
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.02 animals / mL

TEST MEDIUM / WATER PARAMETERS
A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211. The general properties of this medium are as follows.
Total hardness: 2.20 – 3.20 mmol/L
Acid capacity up to pH 4.3: 0.80 – 1.00 mmol/L
Molar ratio Ca:Mg: about 4 : 1
pH value: 7.5 – 8.5
Conductivity: 550 - 650 μS/cm
Total organic carbon: < 2 mg/L
Dissolved oxygen: Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 714 – 762 lux at a wave length of 400 -750 nm. The light intensity will not exceed 1000 – 1500 lux for cool white light (equivalent to 15-20 μE•m-2•s-1 according to the test guideline

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed
every day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each parent for the duration of the study. Separating neonates from adults was accomplished by gently removing adult daphnids from each chamber by means of a wide bore pipet and pouring remaining water and young daphnids through a fine mesh screen. The young collected on the screen were placed in a petri plate and counted on a light table before being discarded. On non-renewal days, the old test solution was collected in a clean beaker and returned to the test vessel along with the parent daphnid. On renewal days, the old test solution was collected from all test group replicates and sampled as required for concentration control analysis when analytical measurements were done. On these days the parent daphnids were returned to their test vessels with fresh test solution.
On day 21 of exposure, some replicates had not produced a complete final brood. Therefore the test was prolonged an extra day to allow Daphnia in all replicates to release their final brood. At test termination, the surviving adults were removed from the test vessels and euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the
helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily. The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

RANGE-FINDING STUDY
In a first experiment, after 21 days exposure to the test substance, a small but statistically significant (p≤0.05) reduction of reproduction was observed in a limit test with a loading rate of 10 mg/L. However this statistical significance may have been due to low variability among the control replicates because reproduction in some replicates of the 10 mg/L treatment exceeded the mean control value (results archived with the raw data from this study). Since the results of the first experiment were inconclusive, a second experiment was conducted using 5 concentrations according to guideline recommendations. In the second experiment all loading rates are above the water solubility, which should conclusively demonstrate biologically relevant effects in a saturated solution of test substance.
Reference substance (positive control):
yes
Remarks:
sodium chloride
Duration:
22 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
reproduction
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
reproduction
Details on results:
BIOLOGICAL RESULTS
Mortality was observed in the highest treatment group (1 parent animal out of 10), however it was not statistically significant and did not follow a concentration response pattern (negative Cochran-Armitage test). The observed mortalities were either accidental or inadvertent. The reproduction endpoint is based on offspring per surviving adult, since there was no concentration response pattern to the mortality.
No additional adverse effects or abnormal behavior were observed in any of the test groups.
No significant mortality, reduced reproduction or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.

DISSOLUTION BEHAVIOR
All test solutions were visibly colorless and clear throughout each renewal period. No undissolved test substance or other unusual manifestations were observed.

ANALYTICAL CONCENTRATION CONTROL
The test substance was determined in the samples by quantifying two ion peaks. No test substance peaks were measured in any of the control samples over the exposure period. All treatment groups were prepared using loading rates above the reported water solubility (0.019 mg/L) to generate saturated solutions of the test substance and it was expected that measured concentrations would be comparable across treatment groups. Peaks corresponding to the test substance were measured in many of the samples from the treatments groups, but many others were below the wLOQ. Results were inconsistent and varied widely both within and between treatment groups. There was no pattern of increasing measured concentration corresponding to the loading rate. The measured concentrations do confirm the presence of the test substance in the test vessels, however they are not suitable to derive exposure levels due to the high variability.
Although the saturated test solutions were filtered to remove undissolved test substance, it is possible that trace amounts of the undissolved test substance passed through the filter which may explain the variability in the measured values. No undissolved test substance was visually observed in the test solutions (see chapter 4.3); however it is impossible to see the trace amounts which were measured.
Since all reasonable efforts were taken to produce saturated solutions of the test substance in test media, following the guidance in OECD 23, the test results should be based on loading rate.
Results with reference substance (positive control):
In order to verify that the Daphnia magna culture is responding normally to toxic stress, acute toxicity tests with a reference
substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline
and in accordance with GLP, but without a GLP status.
The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.90 g/L (experiment date: 11 Jun 2014).
This result is within the range of 3.88 – 7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.
Validity criteria fulfilled:
yes
Conclusions:
Based on the long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic invertebrates.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study according to OECD 211
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is poorly water soluble; therefore a saturated stock solution was prepared following general guidance provided in OECD 23. The substance was first crushed with a mortar and pestle to facilitate accurate weighing. The saturated stock solution was prepared by directly adding 3 mg of test substance to 2L of test medium (1.5 mg/L, loading) and stirred for approximately 1 day.
Prior to toxicity testing, each solution was filtered (Schott glass filtration unit with 0.2 μm nylon membrane, Whatman) to assure the removal of any undissolved test substance. Filters were first wetted and rinsed with at least 100 mL of highly distilled water. Once wet, filters were not allowed to dry out, so some residual water was left standing over the filter. Approximately 500 mL of the test solution was then added to the filtration unit and the first 50 – 90 mL were discarded in order to saturate the filter. The remainder of the test solution was bulk filtered to the point where approximately 10 mL remained standing over the filter. The filtered test solution was used for toxicity testing. Control test media was similarly filtered.
The exposure was started after separation of the undissolved material. The aqueous fraction of the test solution, after separation of the undissolved material, was considered 100% saturated in test media. According to OECD 23, test solutions of poorly soluble substances separated from the remaining undissolved fraction are considered equivalent to the nominal or loading concentration used to prepare them.
Although there was no evidence to suggest exposure concentration instability, fresh test solutions were prepared daily as a precautionary measure to help maintain consistent exposure to the test substance.
All test solutions were visibly colorless and clear throughout each renewal period. No undissolved test substance or other unusual manifestations were observed.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique
Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.
- Age of parental stock (mean and range, SD):
- Feeding during test: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1690 mg TOC/L and 1760 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤118 μL) of the concentrate to reach the amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4- 8°C) for maximum 21 days. By adding the algal concentrate the test solution was slightly diluted. During a 24 h test interval with 1 feeding, a total of 0.118 mL was added to 50 mL test volume resulting in a maximal dilution of 0.2%.
- Feeding schedule:
Day of Test Amount of food per parent animal and day [mg TOC] *
0-3 0.10
4-5 0.12
6-7 0.14
8-21 0.20
*amount of feed algae determined as total organic carbon.

ACCLIMATION
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
2.20 – 3.20 mmol/L
Test temperature:
20 ±1°C
pH:
7.5 – 8.5
Dissolved oxygen:
≥ 3mg/L
Nominal and measured concentrations:
Nominal: 1.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation (test volume: 50 mL)
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.02 animals / mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes.
- Intervals of water quality measurement: The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h darkness
- Light intensity: About 724-764 lux at a wave length of 400-750 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each parent for the duration of the study. Separating neonates from adults was accomplished by gently removing adult daphnids from each chamber by means of a wide bore pipet and pouring remaining water and young daphnids through a fine mesh screen. The young collected on the screen were placed in a petri plate and counted on a light table before being discarded. On each day of the test the parent daphnids were returned to their test vessels with fresh test solution.
On day 21 of exposure, the surviving adults were removed from the test vessels and euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: saturated solution
Basis for effect:
reproduction
Remarks on result:
other: No effects on reproduction and mortality within the range of solubility.
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: saturated solution
Basis for effect:
reproduction
Remarks on result:
other: No effects on reproduction and mortality within the range of solubility.
Details on results:
No additional adverse effects or abnormal behavior were observed in any of the test groups.

Tab 1: Mortality and Reproduction Summary after 21 d

Test Groups
Nominal
 [mg/L]

Reproduction

Mortality

Mean Living Young
per surviving adult

% effectb

Parent animals

% effectb

0 (control)

103 (8.2%a)

0

1.5

108

8

*: p0.05; **: p0.01

a: Coefficient of variation for control fecundity based on surviving parent.

b: Effect relative to control. Only calculated for statistically significant effects.

Two mortalities were observed in the limit test group (20 % mortality) after 10 and 15 days respectively but this was not statistically significant. These individuals did not have reduced reproduction or other signs of toxicity. This minor level of mortality is within the accepted tolerance for control performance (OECD 211) and was thus not toxicologically significant.

Tab. 2: Other biological observations among surviving parent animals in each concentration after a 21 day exposure:

Test Groups
Nominal/TWM
 [mg/L]

Mean Growth
(length, mm)

% Immobile Young

Mean Days to First Brood

% Aborted Eggs

0 (control)

4.17

0.4

9.2

0.6

1.5

4.22

0.0

9.0

0.0

*: p0.05; **: p0.01

-Not measured (100% mortality)

Validity criteria fulfilled:
yes
Conclusions:
Based on long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic organisms.

Description of key information

Based on the long-term (chronic) toxicity study data, the test substance is very likely not harmful to aquatic invertebrates.

Key value for chemical safety assessment

Additional information

Two studies on long-term toxicity to aquatic invertebrates are available for two analogous substances (EC 411-380-6 and EC 410-560-1). Both tests were conducted according to OECD 211 and GLP.

Due to the low solubility of the test substance the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a saturated solution (water saturated fraction, WSF) of the test substance. The test solutions were weighed in and prepared separately (differential loading).

For EC 411-380-6: After 21 d of exposure no significant mortality, reduced reproduction or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. Therefore, the NOEC and the LOEC are determined to be above 1.5 mg/L (saturated solution).

For EC 410-560-1: After 22 d no significant mortality, reduced reproduction or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. Therefore, the LOEC was determined to be > 10 mg/L (loading rate) and the NOEC was determined to be ≥ 10 mg/L (loading rate).

Therefore, the test substance is very likely not harmful to aquatic invertebrates.