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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:
12 December 2000 - 10 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: 1.5 mL from each beaker of every concentration, from fresh solutions of day 0, 6 and 13, and the corresponding aged solutions.
- Sample storage conditions before analysis: no data (presumably analysis was performed on the day of sampling).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was prepared in test dilution water and a measured aliquot of this stock solution was added to test water to prepare the test solutions.
- Eluate: not relevant
- Differential loading: not relevant
- Controls: dilution water only was used as the control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not relevant
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not relevant
- Evidence of undissolved material (e.g. precipitate, surface film, etc): It was confirmed that the test solutions were colourless, clear and had no precipitation.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: no data
- Justification for species other than prescribed by test guideline: not relevant
- Source: under passage breeding at test laboratory; originally obtained from National Institue for Environmental Studies on 15 November 1995
- Age of parental stock (mean and range, SD): no data
- Feeding during test: yes
- Food type: Chlorella vulgaris
- Amount: 0.1-0.2 mg C per daphnid per day
- Frequency: no data

ACCLIMATION
- Acclimation period: 3 weeks
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Chlorella vulgaris, 0.1-0.2 mg C per daphnid per day
- Feeding frequency: no data
- Health during acclimation (any mortality observed): 0% mortality

QUARANTINE (wild caught)
- Duration: not relevant
- Health/mortality: not relevant

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: three weeks and one day prior to the test, matured females bearing offspring were selected from a batch that had 0% mortality during the 29 days before selection and that did not produce diapause eggs or male offspring. Offsprings produced on the next day (within 24 hours) were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
241-258 mg/L
Test temperature:
20-20.8 ºC
pH:
6.7-7.7 (the pH increased with increasing test substance concentration)
Dissolved oxygen:
7.4-9.1 mg/L
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal test concentrations were 0.08, 0.14, 0.26, 0.46, 0.83 and 1.50 mg/L. Concentrations in fresh test solutions were 78.7-121.4% of nominals, and in aged test solutions 72.7-112.5%. As the measured values were outside of the 80-120% range, time-weighted average measured concentrations were used for calculation of the results. For nominal concentrations of 0.08, 0.14, 0.26, 0.46, 0.83 and 1.50 mg/L, respectively, time-weighted average measured concentrations were 0.08, 0.13, 0.24, 0.38, 0.67 and 1.25 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass vessel
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 80 mL test solution per vessel
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not relevant
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): ten
- No. of vessels per control (replicates): ten
- No. of vessels per vehicle control (replicates): not relevant
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: medium according to OECD 211 (M4 medium).
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Salinity: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: pH, temperature and oxygen levels were measured on day 0, 6 and 13 in fresh solutions and also in the corresponding aged solutions.
Other: A full description of the M4 medium in terms of its constituents was provided in the report. The lack of data on TOC and contaminants is considered to be not relevant, since reproductive performance in the control was excellent and the test results were based on analytically confirmed concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 hours dark
- Light intensity: room light (no further data)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): parent daphnia were observed daily for mortality and condition. The number of living offspring was counted daily and the offspring counted were removed. Observations for dead offspring, aborted eggs and diapauses eggs were also recorded daily.

VEHICLE CONTROL PERFORMED: not relevant

RANGE-FINDING STUDY
- Test concentrations: The study design was based on the results of an acute immobilization test. The results of this test were not reported.
- Results used to determine the conditions for the definitive study: no data
Reference substance (positive control):
yes
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
1.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
of parental daphnids
Remarks on result:
other: 0.85-4.07 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 0.63-0.79 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: cumulative number of living juveniles produced per adult alive for 21 days
Remarks on result:
other: LOEC 0.38 mg/L
Details on results:
See table below.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (below mean value of 1.5 mg/L from appendix II of EEC C.2 (acute toxicity to Daphnia magna).
- Relevant effect levels: 48-h EC50 of potassium dichromate 0.90 mg/L
- Other:
Reported statistics and error estimates:
To determine the NOEC and LOEC for reproduction, the Bartlett homoscedastic test and one-way analysis of variance were applied to the cumulative number of living offspring produced per living parent animal during 21 days. The LC50 was determined by the moving average method, and the EC50 for reproduction by logit analysis.

There was no development of diapause eggs or other problems in the control and each test concentration. Other results are summarized in the table below.

 

Table 1. Summary of results of 21-day reproduction test with Daphnia magna

 

nominal concentration

mean meas. TWA concentration

% mortality adults

mean time of first brood (d)

mean no. of offspring per adult alive (day 21)

0

0

0

9.0

185.4

0.08

0.08

20

9.3

174.4

0.14

0.13

0

9.4

176.5

0.26

0.24

0

8.9

186.1

0.46

0.38

0

10.3

121.8*

0.83

0.67

10

11.9

66.9*

1.50

1.25

60

12.5

71.5*

* Significant difference from control (α=0.01)

Validity criteria fulfilled:
yes
Conclusions:
21-day LC50 1.12 mg/L, 21-day EC50 0.70 mg/L, 21-day NOEC 0.24 mg/L..
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the vehicle control and untreated control
- Sampling method: one replicate from each treatment, from fresh solutions of day 0, 1, 8 and 15, and aged solutions of day 7 and 14.
- Sample storage conditions before analysis: no data (presumably analysis was performed on the day of sampling).

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions in DMF were prepared. An aliquot of 0.34 mL of stock solution was added to 3400 mL of test water to prepare the test solutions.
- Eluate: not relevant
- Differential loading: not relevant
- Controls: vehicle controls (0.1 mL/L DMF) and dilution water control were included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): It was confirmed that the test solutions were colourless and transparent at the time of preparation. At the time of replacement, the solution had changed to light green due to the chlorella feed.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Clone A
- Justification for species other than prescribed by test guideline: not relevant
- Source: under passage breeding at test laboratory; originally obtained from the University of Sheffield
- Age of parental stock (mean and range, SD): no data
- Feeding during test: yes
- Food type: Chlorella vulgaris
- Amount: 0.1-0.2 mg C per daphnid per day
- Frequency: no data

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Chlorella vulgaris, 0.1-0.2 mg C per daphnid per day
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no mortality

QUARANTINE (wild caught)
- Duration: not relevant
- Health/mortality: not relevant

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: adults giving birth (20 days of age) were selected, that were raised for more than 14 days under study conditions. During this period this batch had 0% mortality. Offsprings born within 24 hours were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
not relevant
Hardness:
42.7-45.3 mg CaCO3/L
Test temperature:
20.0-20.3°C
pH:
7.3-7.9
Dissolved oxygen:
8.3-9.0 mg/L
Salinity:
not relevant
Nominal and measured concentrations:
Nominal test concentrations were 0.0313, 0.0625, 0.125, 0.25 and 0.500 mg/L. Concentrations in fresh test solutions were 81.1-108% of nominals, and in aged test solutions 63.1-91.9%. As the measured values were outside of the 80-120% range, geometric mean measured concentrations were used for calculation of the results. For nominal concentrations of 0.0313, 0.0625, 0.125, 0.25 and 0.500 mg/L, respectively, geometric mean measured concentrations were 0.0251, 0.0510, 0.107, 0.226 and 0.471 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass container
- Type (delete if not applicable): cylindrical vessel, 9 cm diameter. 12 cm high
- Material, size, headspace, fill volume: max capacity 730 mL, fill volume stated to be 730 mL, headspace not reported.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not relevant
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated water
- Total organic carbon: 0.2 mg/L
- Particulate matter: <1 mg/L
- Metals: an analytical certificate of the dilution water was provided in the report with analysis results of 14 heavy metals. The levels were all below the limit of detection except for aluminium (0.033 mg/L).
- Pesticides: an analytical certificate of the dilution water was provided in the report with analysis results of 12 pesticides or environmental contaminants. The levels were all below the limit of detection.
- Chlorine: <0.02 mg/L
- Alkalinity: 35 mg/L
- Ca/mg ratio: Ca 11.5 mg/L, Mg 3.2 mg/L.
- Conductivity: 18.3 mS/m
- Salinity: not relevant
- Culture medium different from test medium: no
- Intervals of water quality measurement: the analytical certificate was obtained from water sampled on January 9, 2007. The test was initiated on January 19, 2007.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 hours dark
- Light intensity: 15 µE/m2/s
Other: pH, temperature and oxygen levels were measured in fresh solutions of day 0, 1, 8 and 15, and aged solutions of day 7 and 14.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): parent daphnia were observed daily for mortality and condition. The number of living offspring was counted daily and the offspring counted were removed. Observations of size and mobility of parent animals and the presence of resting or aborted eggs were also recorded daily.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: The study design was based on the results of 2 preliminary studies, one performed at concentrations of 0.032, 0.08, 0.2 an 0.5 mg/L, the other one at concentrations of 0.032 and 0.08 mg/L (both studies 5 daphnia/container, 2 containers per treatment and vehicle and untreated control, 15-day duration).
- Results used to determine the conditions for the definitive study: 1st preliminary study: 0% mortality, statistically significant reduction of number of offspring per parent alive after 15 days at 0.5 mg/L, no effects at lower concentrations. 2nd preliminary study: 0% mortality, no effects on number of offspring per parent alive after 15 days at both concentrations. No evidence of hormesis.
Reference substance (positive control):
yes
Remarks:
Acute immobilization test with potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.226 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.403 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.401 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI 0.338-0.532 mg/L
Details on results:
See table below.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (below mean value of 1.5 mg/L from appendix II of EEC C.2 (acute toxicity to Daphnia magna), and also within designated range (mean ± 2 SD) of background data from test laboratory: 0.121-0.349 mg/L).
- Relevant effect levels: 48-h EC50 of potassium dichromate 0.283 mg/L
- Other:
Reported statistics and error estimates:
The reporductive performance was caluated for every 1-day interval bu dividing teh number of offspring by the based on the To determine the NOEC and LOEC for reproduction, the Bartlett test for equality of variances, one-way analysis of variance and Dunnett’s multiple comparison test were applied to the cumulative number of living offspring produced per living parent animals during 21 days. For testing significant differences, the solvent control was used as the control group. No statistically significant difference (p<0.05) was observed between the control and the solvent control. The LC50 was determined by the moving average method, and the EC50 for reproduction by the binomial method.

No growth inhibitions or symptoms were observed in adult daphnia, except at 0.471 mg/L (growth inhibition, immobilization, decreased activity, pale appearance). No resting eggs were formed, but aborted eggs were observed. Dead daphnia magna juveniles were also seen in all cultures except at 0.226 mg/L. There was no development of diapause eggs or other problems in the control and each test concentration. Other results are summarized in the table below.

 

Table 1. Summary of results of 21-day reproduction test with Daphnia magna

nominal concentration

mean meas. TWA concentration

% mortality adults

mean time of first brood (d)

mean no. of offspring per adult alive (day 21)

0 (untreated)

0 (untreated)

0

8.0

80.7

0 (vehicle)

0 (vehicle)

0

8.0

75.0

0.0313

0.0251

0

8.0

86.4

0.0625

0.0510

5

8.0

87.2

0.125

0.107

0

8.0

108**

0.250

0.226

5

8.0

165**

0.500

0.471

65**

8.0

49.0*

** Significant difference from control (α=0.01)

* Significant difference from control (α=0.05)

 

The author of the report qualified the increased reproduction rate at 0.107 and 0.226 mg/L as hormesis. This finding could also be an indication of endocrine disruptive activity. The increased reproductive rate at 0.107 and 0.226 mg/L in the main test (conducted with 4 replicates of 5 daphnia each) was apparent from the start of production of offspring onwards (day 8), and was sustained until day 21. This finding was however not confirmed in the two preliminary tests, conducted with 2 replicates of 5 daphnia each. In the first preliminary test, the number of juveniles per parent alive was 31.2 and 29.5 at 0.08 and 0.2 mg/L , versus 25.6 in the untreated control and 36.7 in the solvent control. In the second preliminary test, the number of juveniles per parent alive was 30.9 at 0.08 mg/L, versus 30.2 in the untreated control and 28.2 in the solvent control. Therefore the increased reproduction rate at 0.107 and 0.226 mg/L observed in the main test is considered to be an incidental finding and not attributable to endocrine disruptive effects. The NOEC proposed in the report of 0.226 mg/L is therefore supported.

Validity criteria fulfilled:
yes
Conclusions:
21-day LC50 0.401 mg/L, 21-day EC50 0.403 mg/L, 21-day NOEC 0.226 mg/L.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The endpoint of long-term toxicity to aquatic invertebrates of the target substance, reaction mass of sodium methyl 2-sulphooctadecanoate and sodium 1-methoxy-1-oxohexadecane-2-sulphonate, was based on the data from the two main components, C16MES and C18MES, and substances similar to the target substance as source chemicals. No definitive value for the reaction mass was derived nor necessary as the data of the two components of the reaction mass are used in separate environmental risk assessments.
Please see the read-across justification in section 13.2
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance: C16MES
Basis for effect:
other: cumulative number of living juveniles produced per adult alive for 21 days
Remarks on result:
other: LOEC 0.38 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance: C16MES
Basis for effect:
reproduction
Remarks on result:
other: 0.63-0.76 mg/L
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
1.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance C16MES
Basis for effect:
mortality
Remarks:
of parental daphnids
Remarks on result:
other: 0.85-4.07 mg/L
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance: C14MES (223-675-5)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
6.81 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance: C14MES (223-675-5)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 8.47 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks:
Substance: C14MES (223-675-5)
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.226 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
Substance: C18MES (223-770-1)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.403 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
Substance C18MES (223-770-1)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.401 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
Substance: C18MES (223-770-1)
Basis for effect:
mortality
Remarks on result:
other: 95% CI 0.338-0.532 mg/L
Conclusions:
The endpoint of long-term toxicity to aquatic invertebrates of the target substance, reaction mass of sodium methyl 2-sulphooctadecanoate and sodium 1-methoxy-1-oxohexadecane-2-sulphonate, was based on the data from the two main components, C16MES and C18MES, and substances similar to the target substance as source chemicals. No definitive value for the reaction mass was derived nor necessary as the data of the two components of the reaction mass are used in separate environmental risk assessments.

Description of key information

C16MES: 21d LC50 = 1.12 mg/L; 21 d repro EC50 = 0.7 mg/L; 21 d repro NOEC = 0.24 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.24 mg/L

Additional information

Data on a similar substance with lower chain length:

C14 MES: 21 d LC50 > 8.47 mg/L; 21 d repro EC50 = 6.81 mg/L; 21 d repro NOEC = 4.10 mg/L

The combination toxicology cannot be used to determine one single key value for chemical safety assessment of the reaction mass - indeed, the proportion of both components that enter the environment (after the STP) is not deemed to be the same as the proportion of the 2 components before the STP