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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:
17 March 2010 to 1 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(see "Principles of method if other than guideline")
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
yes
Remarks:
(see "Principles of method if other than guideline")
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess of test material in reconstituted water for a period of 48 hours prior to removing any undissolved test material present by filtration to give a saturated solution of the test material. Furthermore, the test organisms were Ceriodaphnia dubai rather than Daphnia magna. And the test duration was 8 days rather than 21 days.
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspection date: 15/09/2009 Signature date: 26/11/2009
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of the test material in the test preparations was verified by chemical analysis on Days 0 (fresh media), 2, 5, 7 (old and fresh media) and 8 (old media).
Based on the results of an acute toxicity test to Daphnia magna (Harlan Laboratories Ltd Project Number: 2702/0181) the following test concentrations were assigned to the definitive test: 10, 32 and 100% v/v saturated solution.

- Sampling method: Water samples were taken from the control and each surviving test group for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 2, 5 and 7 and of the expired test preparations on Days 2, 5, 7 and 8.

- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (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 material 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. Aliquots (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: Ceriodaphnia 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 hours 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 was fed 3 to 5 µL of algal suspension 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
Limit test:
no
Total exposure duration:
8 d
Hardness:
140 and 242 mg/L as CaCO3
Test temperature:
25 °C
pH:
7.7 - 8.2
Dissolved oxygen:
7.8 - 8.6 mg/L
Nominal and measured concentrations:
10, 32 and 100% v/v saturated solution - Nominal concentration
0.233 and 0.810 on Day 0 and 0.138 and 0.666 mg/L on Day 2. (as test material) - mean measured concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass vessel
- Type (delete if not applicable): closed (covered to reduce evaporation)
- fill volume: 15 mL of the test preparation
- Aeration: No
- Renewal rate of test solution (frequency): The test preparations were renewed 3 times on Days 2, 5, 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: 43 and 42 mg CaCO3/L in the control and the 100% v/v saturated solution test group at the start of the test.
- Conductivity: 800 and 793 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: 524 to 547 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On a daily basis the numbers 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 general 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 (i.e. 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: 2702/0181) the following test concentrations were assigned to the definitive test: 10, 32 and 100% 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.:
32 other: v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: No live young were produced during the test.
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
10 other: v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: No significant differences (P ≥0.05) between the control and the 10% v/v saturated solution test group in terms of numbers of live young produced per adult by Day 8.
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
immobilisation
Remarks on result:
other: 95 % Confidence Limits Not reported
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
32 other: v/v Saturated solution
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
reproduction
Remarks on result:
other: With 95% confidence limits of 20 - 50% v/v saturated solution
Details on results:
- Mortality of parent animals:
Mortality was observed at the test concentrations of 32 and 100% v/v saturated solution. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 32 and 100% v/v saturated solution test group was not significantly different (P ≥ 0.05) when compared to the control group.
However, no further mortality occurred throughout the test and, hence, no prolonged effects attributable to exposure of Ceriodaphnia dubia to the test material were observed.

- Body length and weight of parent animals:
There was no significant effect on the size and colour of the daphnids at any of the test concentrations compared to the control animals.

- No. of offspring produced per day per female: See table 1, 6 & 7 for details

- Number of males and females (parental): See table 1

- Other biological observations: Sub-lethal effects on the parental generation (P1):
After 8 days there were no statistically significant differences between the control and 10% v/v saturated solution test group in terms of the number of live young produced per adult. The 32% and 100% v/v saturated solution test groups showed a statistically significant difference from the control and the remaining test groups after 8 days in terms of producing fewer numbers of live young per adult.

Effects on the filial generation:
An assessment made at each media renewal showed the "filial" daphnids 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.
Reported statistics and error estimates:
See Any other information on material and methods incl.tables for details.

The following EC50(immobilisation) values based on nominal test concentrations were estimated throughout the test: 

Time

EC50
(% v/v Saturated Solution)

24 hours

>100

48 hours

>100

96 hours

>100

8 days

>100

 

Table 1. Summary of findings following the exposure of Ceriodaphnia dubia for 8 days

Mean Measured Concentration (% v/v Saturated Solution)

% 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

100

248

25

0

0

0

0

10

100

212

21

0

0

0

0

32

70

66

9

0

0

0

0

100

70

55

8

2

<1

0

0

 

*The number of live young per live adult

Table 2. 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

9

0

0

0

3

10

10

5

0

0

4

10

10

22

0

0

5

10

10

52

0

0

6

10

10

67

0

0

7

10

10

49

0

0

8

10

10

53

0

0

 

Total

248

0

0

  

Table 3. Summary of Observations of the 10% v/v Saturated Solution 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

4

0

0

0

3

9

5

0

0

0

4

7

7

4

0

0

5

7

7

15

0

0

6

7

7

5

0

0

7

7

7

21

0

0

8

7

7

21

0

0

 

Total

66

0

0

 

Table 4. Summary of Observations of the 32 % v/v Saturated Solution 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

4

0

0

0

3

9

5

0

0

0

4

7

7

4

0

0

5

7

7

15

0

0

6

7

7

5

0

0

7

7

7

21

0

0

8

7

7

21

0

0

 

Total

66

0

0

 

Table 5. Summary of Observations of the 100 % v/v Saturated Solution 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

4

0

0

0

3

9

7

4

0

0

4

8

6

13

0

0

5

8

6

7

0

0

6

8

7

20

0

0

7

8

7

25

0

0

8

7

6

17

0

0

 

Total

86

0

0

 

Table 6. Total Cumulative Production of Live Young

Mean Measured Concentration (mg/L as test material)

Day

1

2

3

4

5

6

7

8

Control

0

0

5

27

79

146

195

248

10

0

0

5

21

50

86

139

212

32

0

0

0

4

19

24

45

66

100

0

0

4

17

24

44

69

86

 

Table 7. Number of live young produced per adult (Non-cumulative)

Mean Measured Concentration (mg/L as test material)

Day

1

2

3

4

5

6

7

8

Control

0

0

1

2

5

7

5

5

10

0

0

1

2

3

4

5

7

32

0

0

0

0

2

1

3

3

100

0

0

0

1

1

3

3

2

Validity criteria fulfilled:
yes
Remarks:
See Executive summary for details.
Conclusions:
Exposure of Ceriodaphnia dubia to the test material resulted in no significant mortalities at all the test concentrations employed during the test.
The 8-Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Ceriodaphnia generation (P1) was greater than 100% v/v saturated solution.
The 8-Day EC50 (reproduction) based on nominal test concentrations was 32% v/v saturated solution with 95% confidence limits of 20 - 50% v/v saturated solution.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 32% and 10% v/v saturated solution respectively.
Executive summary:

The effect of the test material on the survival and reproduction of Ceriodaphnia dubia over an 8 -day period was investigated in a study which was conducted under GLP conditions and partly in accordance with the standardised guidelines OECD 211 and EPA OPPTS 850.1300.

As the test material is considered to be poorly soluble in water saturated solutions of test material were prepared for use in the study.

Based an acute toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 2702/0181), Ceriodaphnia dubia were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of test concentrations of 10, 32 and 100% v/v saturated solution for a period of 8 days. The test material was prepared by stirring an excess of the test material in reconstituted water (Elendt M4) water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 25°C for 48 hours. After stirring, any undissolved test material was removed by filtration through a pre-conditioned 0.2 µm filter to produce the 100% v/v saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. The test solutions were renewed 3 times. The numbers of live and dead adult Ceriodaphnia and young daphnids (live and dead) were determined daily. The Ceriodaphnia were fed daily with an algal suspension and YAT (yeast, alfalfa, trout chow) combination.

The test material contains a theoretical manganese content of 63% w/w. The test samples were analysed for manganese only. Analysis of the test preparations on days 0, 2, 5, 7 and 8 showed measured concentrations of manganese to be less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.050 mg/L) with the exception of the 32% and 100% v/v saturated solution on Days 0 (fresh media) and 2 (old media). Analysis of these samples showed measured concentrations as test material of 0.233 and 0.810 on day 0 and 0.138 and 0.666 mg/L on day 2. Given that the majority of the analyses were less than the LOQ, it was considered justifiable to base the results on the nominal test concentrations only.

The 8-Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Ceriodaphnia generation (P1) was greater than 100% v/v saturated solution. The 8-Day EC50 (reproduction) based on nominal test concentrations was 32% v/v saturated solution with 95% confidence limits of 20 - 50% v/v saturated solution. The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 32% and 10% v/v saturated solution respectively.

 

                                                             

 

Description of key information

NOEC = 10%v/v saturated solution: OECD 211, EPA OPPTS 850.1300, Priestly & Mullee (2010)

Key value for chemical safety assessment

Additional information

The effect of the test material on the survival and reproduction of Ceriodaphnia dubia over an 8 -day period was investigated in a study which was conducted under GLP conditions and partly in accordance with the standardised guidelines OECD 211 and EPA OPPTS 850.1300.

As the test material is considered to be poorly soluble in water saturated solutions of test material were prepared for use in the study.

Based an acute toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 2702/0181), Ceriodaphnia dubia were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of test concentrations of 10, 32 and 100% v/v saturated solution for a period of 8 days. The test material was prepared by stirring an excess of the test material in reconstituted water (Elendt M4) water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 25°C for 48 hours. After stirring, any undissolved test material was removed by filtration through a pre-conditioned 0.2 µm filter to produce the 100% v/v saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. The test solutions were renewed 3 times. The numbers of live and dead adult Ceriodaphnia and young daphnids (live and dead) were determined daily. The Ceriodaphnia were fed daily with an algal suspension and YAT (yeast, alfalfa, trout chow) combination.

The test material contains a theoretical manganese content of 63% w/w. The test samples were analysed for manganese only. Analysis of the test preparations on days 0, 2, 5, 7 and 8 showed measured concentrations of manganese to be less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.050 mg/L) with the exception of the 32% and 100% v/v saturated solution on Days 0 (fresh media) and 2 (old media). Analysis of these samples showed measured concentrations as test material of 0.233 and 0.810 on day 0 and 0.138 and 0.666 mg/L on day 2. Given that the majority of the analyses were less than the LOQ, it was considered justifiable to base the results on the nominal test concentrations only.

The 8-Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Ceriodaphnia generation (P1) was greater than 100% v/v saturated solution.The 8-Day EC50 (reproduction) based on nominal test concentrations was 32% v/v saturated solution with 95% confidence limits of 20 - 50% v/v saturated solution.The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 32% and 10% v/v saturated solution respectively.