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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-02-2010 to 16-03-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and conducted to a current guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Principles of method if other than guideline:
The study was performed to assess the effect of the test item on the survival and reproduction of Ceriodaphnia dubia over an 8-day period. The method followed that described in the OECD guidelines for Testing of Chemicals No 211 (1998) ''Daphnia magna, Reproduction test'' US EPA short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organism (method 1002.0) and OPPTS Ecological effects test guideline, Daphnid chronic toxicity test OPTTS 850.1300.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not reported
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of the test item in the test preparations was verified by chemical analysis on Day 0 (fresh media), 2, 5, 7 (old and fresh media) and 8 (old media).
- Sampling method:The test samples were analysed following addition of nitric acid (2.5 mL per 50 mL of sample).
- Sample storage conditions before analysis:
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (250 mg) was dispersed in 2.5 litres of reconstituted water (Elendt M4) with the aid of magnetic stirring at approximately 100 rpm at approximately 25°C. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman AcroCap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give the 100% v/v saturated solution. Aliqouts (50 and 160 mL) of the 100% v/v saturated solution were each separately diluted in a final volume of 500 ml of reconstituted water (Elendt M4) to give the remainder of the test series of 10 and 32% v/v saturated solution
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name:Water flea
- Justification for species other than prescribed by test guideline: Ceriodapnia dubia is a freshwater invertebrate representative of a wide variety of natural habitats and can therefore be considered as an important non-target organism in freshwater ecosystems.
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD): 24 hous old
- Feeding during test: Each culture was fed daily
- Food type: Fed a suspension of algae (Pseudokirchneriella subcapitata) and YAT (yeast, alfalfa, trout chow) combination. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Amount: Each daphnid recieved 1µl of a unicellular algal culture and 100 µl of a YAT combination.
- Frequency:Daily

ACCLIMATION
- Acclimation conditions: same as test
- Type and amount of food: same as test
- Feeding frequency: Fed daily
Test type:
static
Water media type:
freshwater
Total exposure duration:
8 d
Hardness:
236 and 230 mg/L as CaCO3
Test temperature:
25°C
pH:
8.1-8.4
Dissolved oxygen:
8.1 - 8.8 mg O2/L
94-106% - Dissolved oxygen concentration expressed as a percentage of air saturation value
Salinity:
Not reported
Nominal and measured concentrations:
10, 32 and 100% v/v saturated solution - Nominal concentration
0.40, 1.3 and 4.1 mg/L (as test item) - Mean measured concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): closed. Covered to reduce evaporation
- fill volume: 50 mL
- Aeration: No
- Renewal rate of test solution (frequency): The test preparations were renewed 3 times on Day 2, 3, and 7.
- No. of organisms per vessel:1
- No. of vessels per concentration (replicates):10
- No. of vessels per control (replicates):10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water (Elendt M4) used for the definitive test was the same as that used to maintain the stock animals.
- Alkalinity: 36 and 37 mg CaCO3/ L in the control and the 4.1 mg/L test group at the start of the test.
- Conductivity: 836 and 784 µs/cm
- Intervals of water quality measurement: The water hardness, conductivity and alkalinity measurements were made at the start of the test.Temperature, oxygen concentration, dissolved oxygen percentage and pH was monitored daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 8 days.
- Light intensity: 528 to 578 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On a daily basis the number of live and dead of the ''Parental'' (P1) generation, the numbers of live and dead ''Filial'' (F1) Ceriodaphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the genral condition and size of the parental Ceriodaphnia as compared with the controls. The number of Ceriodaphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Ceriodaphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 10, 32 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Based on the results of an acute toxicity test to Daphnia magna (Harlan Laboratories Ltd Project Number 2702/0143) the following test concentrations were assigned to the definitive test 10, 32, and 100% saturation v/v saturated solution. As toxicity was not expected, the number of test concentrations was reduced from five, as recommended in the test guideline, to three (10, 32 and 100% v/v saturated solution). This was considered not to affect the validity of the study.
Reference substance (positive control):
no
Duration:
8 d
Dose descriptor:
LOEC
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
reproduction
Remarks on result:
other: (P<0.05)
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Mortality(immobilisation)
Remarks on result:
other: Observed in the Parental generation (P1)
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: No significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 8 days.
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
immobilisation
Remarks on result:
other: Parental Ceriodaphnia generation (P1)
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
reproduction
Remarks on result:
other: With 95% confidence limits of 2.2-2.8 mg/L
Details on results:
- Mortality of parent animals: Mortality (immobilisation occurred predominantly at the highest test concentration of 0.41 mg/L resulting in 50% mortality by Day 8 indicating a prolonged toxic effect attributable to exposure of Ceriodaphnia dubia to the test item. Mortality was also observed at the test concentrations of 0.40 and 1.3 mg/L. However, statistical analysis of the mortality data using the corrected Chi-squared statistics (Breslow and Day 1980) showed that the observed mortalities in the 0.40 and 1.3 mg/L test groups were not significantly different (P≥0.05) when compared to the control group.
- Body length and weight of parent animals: The daphnids at all of the test concentrations were observed to be the same size and colour as the control animals.
- No. of offspring produced per day per female: See table 6, 11 & 12 for details
- Number of males and females (parental): See table 6
- Other biological observations: Sub-lethal effects on the parental generation (P1): After 8 days there were no statistically significant differences between the control, 0.40 and 1.3 mg/L test groups in terms of the number of live young produced per adult. The 4.1 mg/L test group showed a statistically significant difference from the control and the remaining test groups after 8 days in terms of producing fewer number of live young per adult.
Effects on the filial generation: An assessment made at each media renewal showed the ''filial'' dapnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.Numbers of unhatched eggs and dead young were zero in all controls and treatment groups surviving to maturation.
Results with reference substance (positive control):
Not reported
Reported statistics and error estimates:
See Any other information on material and methods inclu.tables for details.

Table 5.The following EC50 (immobilisation) values based on mean measured test concentrations as test item were estimated throughout the test:

 

Time

EC50 (mg/l as test item)

24 hours

>4.1

48 hours

>4.1

96 hours

>4.1

8 days

Approximately 4.1*

*Inspection of the immobilisation data showed an EC50 value of approximately 4.1 mg/l as test item. It was not possible to calculate an EC50 value at this time point due to the unsuitable nature of the data.

 

Table 6. Summary of findings following the exposure of Ceriodaphnia dubia for 8 days.

 

Mean Measured Concentration (mg/l as test item)

% Survival of P1

No. of live young

No. of dead young

No. of unhatched young           

Total

Per female (cumulative)

Total

Per female (cumulative)

Total

Per female (cumulative)

Control

80

124

15

0

0

0

0

0.40

90

167

18

0

0

0

0

1.3

80

124

15

0

0

0

0

4.1

50

12

2

0

0

0

0

 

Table 7. Summary of observations of the control group

 

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

6

0

0

0

3

10

10

0

0

0

4

10

10

0

0

0

5

8

8

31

0

0

6

8

8

12

0

0

7

8

8

41

0

0

8

8

8

40

0

0

 

Total

124

0

0

  

Table 8. Summary of observations of the 0.40 mg/l test group

 

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

5

0

0

0

3

10

10

0

0

0

4

10

10

0

0

0

5

10

10

62

0

0

6

10

10

32

0

0

7

9

9

19

0

0

8

9

9

54

0

0

 

Total

167

0

0

 

Table 9. Summary of observations of the 1.3 mg/l test group

 

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

9

0

0

0

3

10

10

0

0

0

4

10

10

0

0

0

5

9

9

63

0

0

6

9

9

17

0

0

7

8

8

15

0

0

8

8

8

29

0

0

 

Total

124

0

0

 

Table 10.Summary of observations of the 4.0 mg/l test group

 

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

2

0

0

0

3

9

9

0

0

0

4

8

8

0

0

0

5

6

6

2

0

0

6

6

6

7

0

0

7

6

6

3

0

0

8

5

5

0

0

0

 

Total

12

0

0

 

Table 11. Total cumulative production of live young.

 

Mean Measure Concentration (mg/l as test item)

Day

1

2

3

4

5

6

7

8

Control

0

0

0

0

31

43

84

124

0.40

0

0

0

0

62

94

113

167

1.3

0

0

0

0

63

80

95

124

4.1

0

0

0

0

2

9

12

12

 

Table 12. Number of live young produced per adult (Non-cumulative).

 

Mean Measure Concentration (mg/l as test item)

Day

1

2

3

4

5

6

7

8

Control

0

0

0

0

3

2

5

5

0.40

0

0

0

0

6

3

2

6

1.3

0

0

0

0

6

2

2

4

4.1

0

0

0

0

0

1

1

0

Validity criteria fulfilled:
yes
Remarks:
See Executive summary for details.
Conclusions:
Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the test concentration of 4.1 mg/L as test item resulting in 50% mortalities by Day 8. The 8-day EC50 (immobilisation) value, based on mean measures test concentrations as test item, for the parental Ceriodaphnia generation (P1) was estimated to be approximately 4.1 mg/L.
The 8-day EC50 (reproduction) based on mean measures test concentrations as test item was 2.5 mg/L with 95% confidence limits 2.2-2.8 mg/L.
The ''Lowest Observed Effect Concentration'' (LOEC) and the ''No Observed Effect Concentration'' (NOEC) based on mean measures test concentrations as test item were 4.1 and 1.3 mg/L respectively.
Executive summary:

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

The study report includes the entire information requirement as appropriate for OECD 211 (Daphnia magna reproduction test) guideline, except for the fact that the test species used is Ceriodaphnia dubia. Therefore the quality criteria of the study have been fulfilled as the study was conducted to guideline and to GLP.

 

The following validation criteria were achieved during the test:

                                                             

 

Required

Actual

a Control mortality

≤ 20%

20%

b Dissolved oxygen

>3 mg O2/L

≥7.7 mg O2/L

c pH (control group)

Deviation ≤1.5

0.2

d Mean number of live young per surviving adult (control group)

≥ 15 after 8 days

15

e Coefficient of variation for control group

≤ 25%

15%

 

Description of key information

Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the test concentration of 4.1 mg/L as test item resulting in 50% mortalities by Day 8. The 8-day EC50 (immobilisation) value, based on mean measured test concentrations as test item, for the parental Ceriodaphnia generation (P1) was estimated to be approximately 4.1 mg/L.

The 8-day EC50 (reproduction) based on mean measures test concentrations as test item was 2.5 mg/L with 95% confidence limits 2.2-2.8 mg/L.

The ''Lowest Observed Effect Concentration'' (LOEC) and the ''No Observed Effect Concentration'' (NOEC) based on mean measured test concentrations as test item were 4.1 and 1.3 mg/L respectively.  

Key value for chemical safety assessment

Additional information

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

The study report includes the entire information requirement as appropriate for OECD 211 (Daphnia magna reproduction test) guideline, except for the fact that the test species used is Ceriodaphnia dubia. Therefore the quality criteria of the study have been fulfilled as the study was conducted to guideline and to GLP.

 

The following validation criteria were achieved during the test:

                                                             

 

Required

Actual

a Control mortality

≤ 20%

20%

b Dissolved oxygen

>3 mg O2/L

≥7.7 mg O2/L

c pH (control group)

Deviation ≤1.5

0.2

d Mean number of live young per surviving adult (control group)

≥ 15 after 8 days

15

e Coefficient of variation for control group

≤ 25%

15%

 

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