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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-15 to 2010-03-23
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 guidelines and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(see principles of method if other than guideline.)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
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.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100% v/v saturated solution test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis
- Sampling method: The test samples were analysed following addition of nitric acid (1.0 mL per 20 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
- Method:
For the purpose of the definitive test the test material was prepared as a saturated solution. An amount of test material (250 mg) was added to 2.5 litres of reconstituted water and stirred using a magnetic stirrer at approximately 100 rpm for 48 hours. After stirring, any undissolved test material was removed by filtration through a 0.2 µm Gelman AcroCap filter (first approximate 500 mL discarded to pre-condition the filter) to give the 100% v/v saturated solution (equivalent to 0.0219 mg/L as test material based on the mean measured test concentration).

- Eluate: Test water - The reconstituted water for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Controls: A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l. An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/L stock solution. An aliquot (50 mL) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 mL to give a 10 mg/L stock solution. Aliquots (16, 28, 50, 90 and 160 mL) of the 10 mg/L stock solution were each separately dispersed in a final volume of 500 mL of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
EXAMPLE:
TEST ORGANISM
- Common name: Water Flea
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: Each culture was fed daily with a suspension of algae (Chlorella sp.).

ACCLIMATION
- Acclimation conditions: Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°C.


- Type and amount of food: Each culture was fed daily with a suspension of algae (Chlorella sp.). The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Health during acclimation: No mortality observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Temperature was maintained at approximately 20.0°C throughout the test.
pH:
pH range for test samples: 7.8-8.3
Dissolved oxygen:
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%.
Dissolved oxygen concentration for test samples: 8.5 - 9.2 mg/L O2
Nominal and measured concentrations:
Range-finding test: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitive test: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.



Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars containing approximately 200 mL of test preparation were used
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 250 mL
- Aeration: Test vessels not aerated.
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS :Reconstituted water.

i) Stock Solutions
a) CaCl2.2H2O: 11.76 g/L
b) MgSO4.7H2O: 4.93 g/L
c) NaHCO3: 2.59 g/L
d) KCl: 0.23 g/L

ii) Preparation
An aliquot (25 mL) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 μS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: The test vessels were maintained in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.


EFFECT PARAMETERS MEASURED
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution.
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 48-Hour EC50 of greater than 100% v/v saturated solution.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.022 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Based on the mean measured test concentration as test item the 48-Hour EC50 was estimated to be greater than 0.0219 mg/l.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The No Observed Effect Concentration was 100% v/v saturated solution
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Based on the mean measured test concentration the No Observed Effect Concentration was 1.6 mg/l.
Details on results:
Range-finding Test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.
No immobilisation was observed at any of the test concentrations employed in the test. Analysis of the 100% v/v saturated solution showed a measured concentration of manganese of 0.0178 mg/L (equivalent to 0.0247 mg/L as test material). Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.

Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution (equivalent to 0.0219 mg/L as test material based on the mean measured test concentration) for a period of 48 hours. Inspection of the immobilisation data gave the following results:

24h EC50 (% v/v Saturated Solution): >0.0219 (EC50 (mg/L as test material))
48h EC50 (% v/v Saturated Solution) >0.0219 (EC50 (mg/L as test material))

The No Observed Effect Concentration after 24 and 48 hours exposure was 100% v/v saturated solution (equivalent to 0.0219 mg/L as test material based on the mean measured test concentration). The No Observed Effect Concentration is based upon zero immobilisation at this concentration
Verification of test concentrations:
The test material contained a theoretical manganese content of 72% w/w. The test samples were analysed for manganese only. Analysis of the fresh test preparations (see Appendix 3) at 0 hours showed measured concentrations of 0.0214 and 0.0215 mg/L as test material. Analysis of the old media at 48 hours showed measured concentrations of 0.0233 and 0.0215 mg/L as test material.
Given that no decline in measured concentration was observed over the test period, the results are based on the mean measured test concentration as test material only. This was calculated to be 0.0219 mg/L.

Observations on test item solubility
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess 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.
Results with reference substance (positive control):
Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2. Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:

24h EC50 (mg/L):0.84 (95% confidence limits 0.72-0.97 mg/L )
48h EC50 (mg/L):0.65 (95% confidence limits 0.58-0.72 mg/L )
The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/L. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/L (sd = 0.20).
Reported statistics and error estimates:
Evaluation of data for the positive control:
The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999). Probit analysis is used where two or more partial responses to exposure are shown. When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Table1: Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(% v/v Saturated Solution)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

0

100

0

0

 

Table 2: Cumulative Immobilisation Data in the Definitive Test

Mean Measured
Concentration
(mg/L as Test Material)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

0.0219

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0


R1– R4= Replicates 1 to 4

Table 3: Cumulative Immobilisation Data in the Positive Control

Nominal
Concentration
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0.56

1

1

2

10

3

2

5

25

1.0

7

7

14

70

10

10

20

100

1.8

10

10

20

100

10

10

20

100

3.2

10

10

20

100

10

10

20

100


R1– R2= Replicates 1 to 2

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. Based on the mean measured test concentration as test material the 48-Hour EC50 was estimated to be greater than 0.0219 mg/L. Correspondingly the No Observed Effect Concentration was 0.0219 mg/L.This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.
Executive summary:

The short term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

As the test material is considered to be poorly soluble in water the study was conducted with a saturated solution of test material.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 20°C under static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in reconstituted water using a magnetic stirrer at approximately 100 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna were exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.


No daphnia immobilisation was observed over the 48 hour test period. The 48-hour EC50 was therefore determined to be greater than 100% v/v saturated solution. This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.

Description of key information

No toxic effects at the limit of solubility of the test material in the test meidum, OECD 202, EU Method C.2, Goodband & Mullee (2010)

Key value for chemical safety assessment

Additional information

The short term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202 and EU Method C.2.

As the test material is considered to be poorly soluble in water the study was conducted with a saturated solution of test material.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 20°C under static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in reconstituted water using a magnetic stirrer at approximately 100 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm filter to give a saturated solution of the test item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna were exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.


No daphnia immobilisation was observed over the 48 hour test period. The 48-hour EC50 was therefore determined to be greater than 100% v/v saturated solution. This study showed that there were no toxic effects at the limit of solubility of the test material in the test medium.