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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
Study period:
From February 03, 1995 to March 31, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)
Deviations:
yes
Remarks:
due to analytical limitations, no concentrations tested could be analysed. Instead a solution of 1 mg/L was used under the same conditions of the test to prove stability of test concentration.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: 50.5% of test substance in water.
Analytical monitoring:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
Strain / Clone: Daphnia magna clone V
Source: Daphnia magna culture kept as a continuous stock at the test facility. Clone V was originally obtained from the RIZA Lelystad, NL

Breeding method: The stock was kept in 3 L beakers containing approximately 2.5 L of culture medium. The maximum number of daphnids per test
vessel was 150 for the animals being 0 to 6 d old and 80 for the animals being older than 6 d. The culture medium was batchwise refreshed at least twice a week. During culturing the animals were fed six times a week with a chlorella-suspension. The daphnid culture was kept at 19-21ºC, 8 h dark and 16 h light and light intensity of approximately 1000 lux.
Kind of food: Chlorella pyrenoidosa suspension
Feeding frequency: Six times a week
Pretreatment: No
Feeding of animals during test: Yes,
Day food (µL)
0 15
1 15
2 20
3 30
4 40
5 50
6 60
7 70
8 80
9 90
10-20 100
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Nominal and measured concentrations:
Nominal: 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg/L
Details on test conditions:
The study was performed according to the draft OECD test guideline 202, part II: Daphnia, reproduction test, draft 6/93 with some modifications which were:
- The composition of the dilution water was according to a correction of the guideline.
- The food amount was not recalculated into TOC-content.
- The number of offspring and the number of parent animals was not counted daily, but on the Days of renewal of the test solutions only.
- The number of test vessels and the number of animals per test vessel.
- The number of offspring was tested in a two-sided Dunnett-test. 20 Daphnia magna per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg/L of the test substance for 22d under semi-static conditions. There was no analytical method available to analyse the very low concentrations used in this test. All values given are therefore nominal concentrations. The number of living offspring per parent animal per d is assessed as is the mortality of the parent animals. A NOEC and LOEC was determined for reproduction and for mortality of the parent animals. In order to prove the stability of the test substance a stability study was performed under test conditions at a concentration of 1 mg/L. Percentage recovery ranged between 98 and 107%.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.013 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
NOEC mortality parent animals = 0.025 mg/L
LOEC mortality parent animals = 0.05  mg/L. 
At the next higher concentration, 0.1 mg/L all parent animals died within 48 h. Therefore there was no significant inhibition nor a stimulation effect observed at all concentrations tested.  The NOEC and LOEC for reproduction therefore amounted to respectively 0.05 mg/L and 0.1 mg/L.
Based on active substance:
NOEC mortality parent animals = 0.0125 mg a.i./L.
LOEC mortality parent animals = 0.025  mg a.i./L. 
NOEC (reproduction) = 0.025 mg a.i./L.
LOEC (reproduction) = 0.05  mg a.i./L. 
- Conclusion: 
No effects on reproduction were observed at the highest level which could be tested. The 48h-NOEC and LOEC are about a factor three higher than the 48h-NOEC and LOEC from the acute daphnia toxicity study. (NOEC 0.0125 vs 0.0048 mg a.i./L; LOEC 0.025 vs 0.0075 mg a.i./L). Although somewhat high, it is considered to be caused by normal biological variation. Besides, the NOEC and LOEC in this study are defined by a statistical significant difference of the test group to the control. Although at 0.006 mg a.i./L cumulative mortality reached 40% compared to 16% in the control, but this did not reach statistical significant level. The 48h-EC100 is in both studies the same: 0.05 mg a.i./L Irrespective of the differences in acute toxicity, this study demonstrates that reproduction capacity only appears subsequent to acute toxicity, and that the chronic NOEL therefore does not differ from the acute NOEL.
Results with reference substance (positive control):
-
Reported statistics and error estimates:
Established on statistical evaluation of difference to controls
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 21d NOEC (mortality parent animals) was 0.0125 mg a.i./L and the LOEC (mortality parent animals) was 0.025 mg a.i./L. At the next higher concentration, 0.05 mg a.i./L, all parent animals died within 48 h. Therefore, there was no significant inhibition nor a stimulation effect observed at all concentrations tested.  The NOEC and LOEC for reproduction therefore amounted to respectively 0.025 mg a.i./L and 0.05 mg a.i./L in Daphnia magna. 
Executive summary:

A study was conducted to determine the long-term toxicity to aquatic invertebrates according to OECD Guideline 211, in compliance with GLP. Twenty daphnids per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg a.i./L of test substance for 21 days under semi-static conditions. There was no analytical method available to analyse the very low concentrations used in the study. All values given were therefore nominal. The number of living offspring per parent per day was assessed, as well as the mortality of the parent animals. The NOEC and LOEC were determined for reproduction and mortality of the parent animals. In order to prove the stability of the test substance, a study was performed under test conditions at a concentration of 1 mg/L. Percentage recovery ranged between 98 and 107%. Under the study conditions, the 21d NOEC (mortality parent animals) was 0.0125 mg a.i./L and the LOEC (mortality parent animals) was 0.025 mg a.i./L. At the next higher concentration, 0.05 mg a.i./L, all parent animals died within 48 h. Therefore, there was no significant inhibition nor a stimulation effect observed at all concentrations tested.  The NOEC and LOEC for reproduction therefore amounted to respectively 0.025 and 0.05 mg a.i./L (Mark, 1995).

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 30, 1990 to November 20, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: U.S. EPA FIFRA 72-4 (b)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyl(C12-C16)dimethylbenzylammonium chloride (ADBAC) (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: Light beige solid
- Analytical purity: 30% w/w a.s. in aqueous solution
- Radiochemical purity (if radiolabelling): 96.5% (by TLC) and 95.5% (by HPLC).
- Locations of the label (if radiolabelling): 14 C carbon of the benzene ring
- Stability under test conditions: The non-radiolabelled a.s., ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage conditions: Under referigeration
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
Dilution water:
The base water used was Millipore Milli-Q™ deionized reverse osmosis treated well water.

Preparation of stock solution:
14C-test substance primary stock solutions were used to prepare test solutions for daphnid exposure. The primary stock solutions of 14C-test substance were prepared by adding measured aliquots of 14C-test substance to measured amounts of deionized water in volumetric glassware followed by shaking to ensure homogeneity. Three separate primary stock solutions were made and used during the course of the study. The 14C-test substance concentrations of the primary stock solutions were verified by liquid scintillation counting (LSC).

Test solution preparation:
Test solutions used in the study were prepared by daily adding measured aliquots of 14C-test substance primary stock solutions to dilution water in 2L volumetric flasks and bringing the volume to 2 L. Prior to adjusting the final volume to 2L, algal cells and a prepared food suspension were added to the dilution water in the 2L volumetric flask. After addition of the stocl solution and food, the test solutions were vigorously mixed to ensure homogeneity and solubilisation of the test substance.
- Test solutions were renewed daily and prepared immediately before use. At renewal, daphnids were transferred by wide-bore pipette from vessels with the 24-h test solution to the appropriate test vessels containing new test solution. In order to verify test substance concentrations in the test solutions for the deifinitive study, triplicate 1 mL aliquots were taken daily from each newly made test solutions (new solution) before transfer to the individual test vessels and from the three replicates from each test solution and control 24 h later at the time of renewal (old solutions) and counted by LSC.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Daphnia magna was originally obtained from U. S. EPA’s Environmental Research Laboratory, Duluth, MN and was cultured in an environmental chamber under controlled conditions (temperature: 20±2°C; photo-period: 16-h light - 8-h dark; light intensity: 50-150 fc).
- Daphnia were cultured in 1L glass beakers (10 Daphnia per beaker) containing 800 mL of hard re-constituted laboratory water.
- Each beaker received 1.8E08 cells/L selenastrum capricornutum and 5 mL of a yeast/trout food/cerophyl suspension three times each week when the culture water was changed. Selenastrum was obtained from Battelle in-house cultures (algal slants originally obtained from the university of Texas at Austin). Purina brand trout chow, Fleischmann brand yeast and Sigma chemical company cerphyl were sources for the YTC suspension.
- Survival and reproduction of culture animals were checked each time the culture water was changed. After 28d, the adults were discarded and new cultures were started.
- 24h before the start of the test, young were removed from the beakers to ensure that only daphnids less than 24-h old would be used to start the test. the young daphnids were from 22d old cultures. There was 100% survival of cultures animals and an average of 8.6 young produced per female per reproductive day the week before the toxicity test.


Test type:
other: daily static renewal without aeration
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
160.5 mg/L as CaCO3 (124 to 228 mg/L)
Test temperature:
18.1 to 22°C
pH:
7.4 to 8.4
Dissolved oxygen:
5.6 to 9.6 mg/L (represents 64to 109% air saturation at 20°C)
Nominal and measured concentrations:
- Nominal concentrations of 0 (dilution water control), 0.5, 0.9, 1.6, 2.8 and 5 µg/L.
- Measured concentration: 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg/L
Details on test conditions:
Test system:
- Test vessel: Ten 250 mL test vessels contained 200 mL of test solution (depth of 60 mm) were used for each concentration and control.
- Number of replicates: Two for control and each test concentration.
- Number of Daphids: Seven of the ten test vessels contained an individual daphnid in each vessel while the three remaining vessels for each test concentration and control contained five daphnids each (total number of daphnids = 22 per test concentration or control).
- Aeration: No

Test conditions:
- Daphnia magna less than 24h old were exposed to mean analytical concentrations of 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg/L of the test substance in a 21d daily static-renewal test without aeration. Each concentration was run in duplicate. Test solutions were renewed daily and prepared immediately before use. Observations on daphnid behaviour, the number of live and dead (or immobilised) organisms, and the number of young produced were made at each renewal day and at test termination.
- Test temperature: 20±2°C
- Photoperiod: 16h light and 8h darkeness
- Light intensity: 103.9 fc

Observations:
- Parameters observed: The number of live and dead (or immobilized) organisms, and the number of young produced were made at each renewal day and at test termination.
- Actual measurements of the test daphnids were made in ocular units and converted to mm (1 ocular unit = 0.07 mm).
- Reproduction and growth (length) data were only collected from the seven individually exposed replicates in each exposure concentration and control. Survival data were collected for all exposure beakers.

Range-finding study:
- A 9 -d range finding experiment with Daphnia magna neonates (<24h old) were exposed to five nominal conentrations 0.1, 0.5, 1, 5 and 10 µg/L of 14C-test substance and a dilution water control.
- Two replicates each were used.
- Five neonates were placed in each test concentration and control vessel (total of 12 beakers and 60 daphnids) with a wide-bore pipette.
- Test vessels were maintained in an environmental chamber at a temperature of 20±2°C. Aeration was not used.
- For the duration of the test, observations were made on survival and reproduction of the test organisms.
- Test substance concentration were verified by using LSC.
- Based on the results from the range finding study, the definitive chronic toxicity was conducted at nominal concentrations of 0.5, 0.9, 1.6, 2.8 and 5 µg/L 14C-test substance.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 4.15 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, reproduction and growth
Details on results:
Range-finding study:
- At test termination survival was ≥90% in all test concentrations and control.
- Total reproduction (live plus dead young) in the nominal 14C-test concentrations of 0 (control), 0.1, 0.5, 1, 5 and 10 µg/L was 42, 51, 58, 52, 0 and 0 young respectively.
- At test termination, a total of 2, 33 and 41 aborted eggs were found in the 1, 5, and 10 µg/L nominal test concentrations respectively.
- The overall mean of analytical values for the 0.1, 0.5, 1, 5 and 10 µg/L nominal concentrations were 0.06, 0.5, 0.96, 5.02 and 9.24 µg/L respectively.

Main study:
- The LC50 values was >4.15µg/L.
- At the end of the 21-d exposure, there was 86% survival of the test organisms in the highest measured test concentration (4.15 µg/L) and 95% or greater survival in the remaining test concentrations and in the dilution water control. Survival in all the test concentrations was not significantly different (p>0.05) from the control.
- The NOEC and LOEC for survival, therefore, were >4.15 and >4.15 µg/L of the test substance respectively.
- Reproduction of the test organisms was not significantly affected (p>0.05) in any of the test concentrations when compared to the control by ANOVA. The NOEC determined from the 21d reproductive data was ≥4.15 µg/L of the test substance.
- The growth of the test organisms at the end of the 21-d exposure period was also not significantly different (p>0.05) in any of the test concentration when compared with control using ANOVA. The NOEC and the LOEC determined from the 21d growth data was ≥4.15 and >4.15 µg/L respectively.
Reported statistics and error estimates:
Fisher’s Exact Test, analysis of variance, Bonferroni’s multiple comparison test.

Table 1. NOEC and LOEC results

Parameter

Concentration (µg/l)

Mortality

NOEC

4.15

LOEC

> 4.15

Reproduction

NOEC

4.15

LOEC

5.02

Growth

NOEC

4.15

LOEC

> 4.15

No treatment related effects were observed in the definitive 21d study which was conducted at nominal test concentrations of 0.5, 0.9, 1.6, 2.8 and 5 µg/L. This result was surprising in light of the fact that marked effects on reproduction were observed at nominal concentrations at or above 5 µg/L in the 9 -day range-finding study.

One possible explanation for these results was that the mean analytical or actual test concentration acheived in the nominal 5 µg/L treatment group for the 9 -day range finding study was 5.02 µg/L, while the analytical test concentration for the definitive study was only 4.15 µg/L. While no effects were observed in the definitive study, the data from the range finding study clearly demonstrates effects on reproduction at test concentrations at or above 5.02 µg/L. In addition, the 48 -h LC50 in daphnids was determined at this laboratory to be 5.8 µg/L. Therefore, while an effect level was not demonstrated in the definitive study, the principle objective of the definitive study was still accomplished since the no-effect levels for the paramters of reproduction and growth were clearly defined at levels just below concentrations which have been shown to produce effects on reproduction and survival.

By supplementing the results of the 21 d definitive test with those from the 9 d range finding test, the following conclusions were drawn:

- The NOEC and LOEC for reproduction were 4.15 µg/L and 5.02 µg/L respectively.

- The MATC derived for the combined test results was 4.56 µg/L.

- The NOEC and LOEC for survival and growth are ≥4.15 and >4.15 µg/L µg/L respectively.

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study, the 21d chronic NOEC for mortality, reproduction and growth (Daphnia magna) was found to be ≥ 4.15 µg a.i./L of test substance. The maximum acceptable toxicant concentration (MATC) was 4.56 µg/L.
Executive summary:

A study was conducted to determine the long-term toxicity to aquatic invertebrates of the test substance according to U.S. EPA FIFRA 72-4 (b), in compliance with GLP. Daphnia magna (22 per group) were exposed to mean measured concentrations of 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg a.i./L for 21 days. Analytical dose verification was conducted. Observations on daphnid behaviour, the number of immobilised organisms and the number of young produced were made at each renewal day and at test termination. No effects were observed on survival, reproduction and growth at measured concentration up to 4.15 µg a.i./L test substance. However, reproduction was clearly affected at measured concentrations ≥ 5.02 µg/L in a range-finding study. Based on these results, the NOEC and LOEC for reproduction were considered to be at 4.15 and 5.02 µg a.i./L, respectively. The maximum acceptable toxicant concentration (MATC) derived from these results was 4.56 µg a.i./L. The NOEC and LOEC for survival and growth were ≥ 4.15 and > 4.15 µg a.i./L, respectively. Under the conditions of the study, the 21d chronic NOEC for mortality, reproduction and growth (Daphnia magna) was found to be ≥ 4.15 µg a.i./L (≥ 0.00415 mg a.i./L) of test substance. The maximum acceptable toxicant concentration (MATC) was 4.56 µg/L (McIntyre, 1992).

Description of key information

Based on effects on reproduction, the 21 d NOEC value forDaphnia magna(freshwater) due to the test substance was determined to be 0.025 mg a.i./L (nominal).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.025 mg/L

Additional information

Study 1. A study was conducted to determine the long-term toxicity to aquatic invertebrates according to OECD Guideline 211, in compliance with GLP. Twenty daphnids per concentration were exposed to 0, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1 or 0.2 mg a.i./L of test substance for 21 days under semi-static conditions. There was no analytical method available to analyse the very low concentrations used in the study. All values given were therefore nominal. The number of living offspring per parent per day was assessed, as well as the mortality of the parent animals. The NOEC and LOEC were determined for reproduction and mortality of the parent animals. In order to prove the stability of the test substance, a study was performed under test conditions at a concentration of 1 mg/L. Percentage recovery ranged between 98 and 107%.Under the study conditions, the 21d NOEC (mortality parent animals) was 0.0125 mg a.i./L and the LOEC (mortality parent animals) was 0.025 mg a.i./L. At the next higher concentration, 0.05 mg a.i./L, all parent animals died within 48 h. Therefore, there was no significant inhibition nor a stimulation effect observed at all concentrations tested.  The NOEC and LOEC for reproduction therefore amounted to respectively 0.025 and 0.05 mg a.i./L (Mark, 1995).

Study 2. A study was conducted to determine the long-term toxicity to aquatic invertebrates of the test substance according to U.S. EPA FIFRA 72-4 (b), in compliance with GLP. Daphnia magna (22 per group) were exposed to mean measured concentrations of 0, 0.41, 0.71, 1.33, 2.31 and 4.15 µg a.i./L for 21 days. Analytical dose verification was conducted. Observations on daphnid behaviour, the number of immobilised organisms and the number of young produced were made at each renewal day and at test termination. No effects were observed on survival, reproduction and growth at measured concentration up to 4.15 µg a.i./L test substance. However, reproduction was clearly affected at measured concentrations ≥ 5.02 µg/L in a range-finding study. Based on these results, the NOEC and LOEC for reproduction were considered to be at 4.15 and 5.02 µg a.i./L, respectively. The maximum acceptable toxicant concentration (MATC) derived from these results was 4.56 µg a.i./L. The NOEC and LOEC for survival and growth were ≥ 4.15 and > 4.15 µg a.i./L, respectively. Under the conditions of the study, the 21d chronic NOEC for mortality, reproduction and growth (Daphnia magna) was found to be ≥ 4.15 µg a.i./L (≥ 0.00415 mg a.i./L) of test substance. The maximum acceptable toxicant concentration (MATC) was 4.56 µg/L (McIntyre, 1992).