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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:
3 March 2010 to 17 March 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 items, 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 Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test items has shown that a mixing period of
24 - 48 hours is sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
Furthermore, the test organisms were Ceriodaphnia dubai rather than Daphnia magna. Moreover, the test duration was 8 days rather than 21 days.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The concentration of the test material in the test preparations was verified by chemical analysis on Day 0 (fresh media), 3, 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: 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: Due to the low aqueous solubility and complex nature of the test material for the purpose of the definitive test the test material was prepared as a water accommodated fraction (WAF).

Amount of test material (2.5, 8.0, 25, 80 and 250 mg) were each separately added to the surface of 2.5 litres of reconstituted water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2, 10, 32, and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no undissolved test material or micro-dispersions to be present.


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 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 received 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
Limit test:
no
Total exposure duration:
8 d
Hardness:
240 mg/L as CaCO3
Test temperature:
25 °C
pH:
8.0-8.8
Dissolved oxygen:
8.0 - 9.4 mg O2/L
83- 110% - Air saturation value. The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in exceaa of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.
Nominal and measured concentrations:
1.0, 3.2, 10, 32 and 100 mg/L - Nominal loading rates
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: 39 and 38 mg CaCO3/ L in the control and the 100 mg/L loading rate WAF test group at the start of the test
- Conductivity: 1250 and 801 µs/cm in the control and the 100 mg/L loading rate WAF test group at the start of the test
- Intervals of water quality measurement: Temperature was recorded, twice in two different locations, and light intensity was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded before and after test media renewal

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 8 days
- Light intensity: 526 to 590 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 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 (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
- 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 2702/0161).
Reference substance (positive control):
no
Duration:
8 d
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: (P<0.05)
Duration:
8 d
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate WAF
Basis for effect:
other: Mortalilty(immobilisation)
Remarks on result:
other: Observed in the Parental generation (P1)
Duration:
8 d
Dose descriptor:
other: EL*50
Effect conc.:
27 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Nominal loading rates
Basis for effect:
immobilisation
Remarks on result:
other: Parental Ceriodaphnia generation (P1). 95% confidence limits of 32-100 mg/L loading rate WAF.
Duration:
8 d
Dose descriptor:
other: EL*50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: With 95% confidence limits of 6.5-20 mg/L loading rate WAF
Details on results:
- Mortality of parent animals:
Mortality (immobilisation) occurred predominantly at the highest loading rate of 100 mg/L resulting in 100% mortality by Day 5 indicating a prolonged toxic effect attributable to exposure of Ceriodaphnia dubia of the test material. Mortalities were also observed at the 1.0, and 3.2 mg/L loading rate WAF test groups. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 1.0 and 3.2 loading rate WAF test groups were not significantly different (P≥0.05) when compared to the control. No mortality occurred at the 10 and 32 mg/L loading rate WAF test group throughout the test (See table 5).

- Body length and weight of parent animals:
There was no significant effect on the size of the daphnids at the 100 mg/L loading rate WAF test group prior to observing 100% mortalities by Day 6. Thedaphnids at the remaining loading rates were observed to be the same size as the control animals.

- Number of males and females (parental):
After 8 days there were no statistically significant differences between the control and the 1.0 and 3.2 mg/L loading rate WAF test groups in terms of the number of live young produced per adult. The 10 and 32 mg/L loading rate WAF groups showed a statistically significant difference from the control after 8 days in terms of producing fewer numbers of live young per adult.
Reported statistics and error estimates:
See "Any other information on material and methods incl.tables" for details.

Table 5: The following EL*50 (immobilisation) values based on nominal loading rates were estimated by inspection of the immobilisation data at 24 an 48 hours and calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) at 96-hours and the geometric mean method at 8 days:

 

Time

EL*50 (mg/L)

95% Confidence Limits (mg/L)

24 hours

>100

-

48 hours

>100

-

96 hours

75

51-110

8 days

57

32-100**

*EL Effective loading rate

** Concentrations resulting in 0 and 100% immobilisation respectively

 

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

Nominal loading rate (mg/L)

% 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

140

17

0

0

0

0

1.0

80

139

17

1

<1

0

0

3.2

90

128

16

4

<1

0

0

10

100

83

8

2

<1

0

0

32

100

30

3

0

0

0

0

100

0

0

0

0

0

0

0

*The number of live and dead young and unhatched eggs per live female remaining on day 8.

 

Table 7: Summary of observations of the control group

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

2

0

0

0

3

10

9

0

0

0

4

9

9

15

0

0

5

9

9

32

0

0

6

9

9

28

0

0

7

9

9

35

0

0

8

8

8

30

0

0

 

Total

140

0

0

 

 

Table 8: Summary of observations of the 1.0 mg/L loading rate WAF test group

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

3

0

0

0

3

10

7

5

0

0

4

9

9

17

0

0

5

8

8

20

0

0

6

8

8

22

0

0

7

8

8

42

0

0

8

8

8

33

0

0

 

Total

132

0

0

 

Table 9: Summary of observations of the 3.2 mg/L loading rate WAF test group

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

1

0

0

0

3

10

8

3

0

0

4

10

9

24

0

0

5

9

9

19

0

0

6

9

9

13

0

0

7

8

10

32

0

0

8

8

9

37

0

0

 

Total

128

0

0

 

Table 10: Summary of observations of the 10.0 mg/L loading rate WAF test group

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

1

0

0

0

3

10

10

0

0

0

4

10

10

12

0

0

5

10

10

11

0

0

6

10

10

21

0

0

7

10

10

13

0

0

8

10

10

26

0

0

 

Total

83

0

0

 

Table 11: Summary of observations of the 32 mg/L loading rate WAF test group

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

0

0

0

0

3

10

7

0

0

0

4

10

10

14

0

0

5

10

10

7

0

0

6

10

10

1

0

0

7

10

10

6

0

0

8

10

10

2

0

0

 

Total

30

0

0

 

Table 12: Summary of observation of the 100 mg/L loading rate WAF test groups. 

Day

Adults Surviving

Number of adults with eggs in brood pouch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

0

0

0

0

3

6

3

0

0

0

4

3

2

0

0

0

5

A/D

 

 

0

0

 

Total

0

0

0

A/D all daphnids dead.

 

Table 13: Total cumulative production of live young

Nominal loading rate (mg/L)

Day

1

2

3

4

5

6

7

8

Control

0

0

0

2

4

3

4

3

1.0

0

0

1

2

2

3

5

4

3.2

0

0

0

2

2

1

3

4

10

0

0

0

1

1

2

1

3

32

0

0

0

1

1

0

1

0

100

0

0

0

0

A/D

 

 

 

A/D all daphnids dead.

 

Table 14: Number of live young produced per adult (non-cumulative)

Nominal loading rate (mg/L)

Day

1

2

3

4

5

6

7

8

Control

0

0

0

15

47

75

110

140

1.0

0

0

5

22

42

64

106

139

3.2

0

0

3

27

46

59

91

128

10

0

0

0

12

23

44

57

83

32

0

0

0

14

21

22

28

30

100

0

0

0

0

A/D

 

 

 

 

Analytical results

The test concentrations were monitored by analysis of the test samples for manganese only.

Analysis of the fresh test preparations on days 0, 3, 5 and 7 showed measured concentrations of manganese to range from 0.144 to 3.49 mg/L. Analysis of the old test preparations on days 3, 5, 7 and 9 showed measured concentrations of manganese to range from less that the limit of quantitation (LOQ) of the analytical method (0.050 mg/L as Mn) to 4.47 mg/L

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

 

  

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Ceriodaphnia dubia to the test material resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100% mortality by Day 6. The 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF.
The 8-Day EL*50(reproduction) based on nomainal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF.
The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF 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 in accordance with the standardised guidelines OECD 211 and EPA OPPTS 850.1300.

During the study, Ceriodaphnia dubai were exposed to Water Accommodated Fractions of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 8 days. The WAFs were renewed 3 times. The numbers of live and dead adult Ceriodaphnia and young daphnids (live and dead) were determined daily.

Under the conditions of the study, the 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF. The 8-day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF. The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF, respectively.

Description of key information

8-day NOEL = 3.2 mg/L loading rate WAF (nominal), 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 in accordance with the standardised guidelines OECD 211 and EPA OPPTS 850.1300.

During the study, Ceriodaphnia dubai were exposed to Water Accommodated Fractions of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 8 days. The WAFs were renewed 3 times. The numbers of live and dead adult Ceriodaphnia and young daphnids (live and dead) were determined daily.

Under the conditions of the study, the 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF. The 8-day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF. The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF, respectively.