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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:
10 February 2010 to 9 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:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
(See principles of method if other than guideline.)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 15/09/09 Date of signature: 26/11/09
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples were taken from the control and each loading rate WAF test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.
- 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:
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) to 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 used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: 1st instar Daphnia magna derived from in-house laboratory cultures
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24 hours old
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: parthenogenesis
- Feeding during test: the daphnids received no food during the test

ACCLIMATION
- Acclimation period: overnight

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
The number of immobilised Daphnia were recorded after 24 and 48 hours.
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3. (see appendix 3 in the attachments for detailed results).
Test temperature:
Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510. The test temperature was maintained at 21 °C throughout the study. (see appendix 3 in the attachments for detailed results).
pH:
The pH values were recorded at the start and termination of the test. The pH concentrations were measured using a WTW pH/Oxi 340I pH meter. Differences in pH were observed at 0 hours between the control and the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. (see appendix 3 in the attachments for detailed results).
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentrations were measured using a
dissolved oxygen meter. There were no treatment related differences for oxygen concentration. (see appendix 3 in the attachments for detailed results).

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 in the control group.
Nominal and measured concentrations:
- Nominal concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L

Measured concentrations:
Analysis of the fresh media at 0 hours showed measured concentrations of manganese to range from 1.13 to 3.79 mg/L. Analysis of the old media at 48 hours showed measured concentrations of manganese to range from less than the limit of 1.16 to 3.62 mg/L.

Analysis of the control samples at 0 and 48 hours showed measured concentrations of 0.0155 and 0.0159 mg manganese/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250mL glass jars
- Type (delete if not applicable): closed top reduce evaporation.
- Material, size, headspace, fill volume: 200 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): no test solution renewable
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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.

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

OTHER TEST CONDITIONS
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results from the range finding study, loading rates of 10, 18, 32, 56 and 100 mg/L, using a stirring period of 47 hours followed by a 1-hour standing period, were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL 41 - 46
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Details on results:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (please see the any other information on results section for all tables). Immobilisation was observed predominately at the 100 mg/L loading rate WAF.

A single immobilised daphnid was observed at the 1.0 and 10 mg/L loading rate WAFs after 48 hours exposure. These were considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed.

Analysis of the 100 mg/L loading rate WAF (see appendix 2) showed a measured concentration of manganese of 2.79 mg/L.

Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L, using a stirring period of 47 hours followed by a 1-hour standing period, were selected for the definitive test.
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 4. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 2 and 3.
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:

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72

The No Observed Effect Concentrations 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:
An estimate of the EL*50 value at 24 hours was given by inspection of the immobilisation data.

The EL*50 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).

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

DefinitiveTest:

Immobilisation data:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. The relationship between percentage immobilisation and loading rate at 48 hours is given in Figure 1.

Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal loading rates gave the following results:

Time (h)

EL*50(mg/L)

95% Confidence limits
(mg/L)

24

> 100**

-

48

43

41 - 46

The No Observed Effect Loading rate after 24 and 48 hours exposure was 32 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

A single immobilised daphnid was observed at the 10 mg/L loading rate WAF after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/L loading rate WAFs.


*EL = Effective Loading rate

** An EL50value could not be calculated as less than 50% immobilisation occurred at this time point

Table 1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate

(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

1[1]

10

0

1*

100

5

10


[1] Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed

Table 2: Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate

(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

10

1[1]

0

1*

5

1*

0

1*

5

18

0

0

0

0

0

0

0

0

32

0

0

0

0

0

0

0

0

56

2

2

4

20

9

10

19

95

100

1

1

2

10

10

10

20

100


R1– R2= Replicates 1 and 2

[1] Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/l loading rate WAFs

Table 3: Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/L)

Control

10

18

*

+

*

+

*

+

Height of Water Column (cm)

~17

~17

~17

~17

~17

~17

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

 

 

Nominal Loading Rate (mg/L)

32

56

100

*

+

*

+

*

+

Height of Water Column (cm)

~17

~17

~17

~17

~17

~17

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present


* Start of mixing period

+ End of mixing period

Table 4: 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

9

19

95

10

10

20

100

3.2

10

10

20

100

10

10

20

100

Analytical results

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

Analysis of the fresh media at 0 hours showed measured concentrations of manganese to range from 1.13 to 3.79 mg/L. Analysis of the old media at 48 hours showed measured concentrations of manganese to range from less than the limit of 1.16 to 3.62 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:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EL*50 value of 43 mg/L loading rate WAF with 95% confidence limits of 41 - 46 mg/L loading rate WAF. The No Observed Effect Loading rate at 48 hours was 32 mg/L loading rate WAF.
Executive summary:

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours under at a temperature of 21°C under static test conditions. The number of immobilised Daphnia 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.

Under the conditions of the test, the 48-hour EL*50 for the test material, based on nominal loading rates, was 43 mg/L loading rate WAF with 95% confidence limits of 41 - 46 mg/L loading rate WAF. The No Observed Effect Loading rate was 32 mg/L loading rate WAF.

The 48-hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.78 mg/L with 95% confidence limits of 0.58 – 0.72 mg/L. The No Observed Effect Concentration was 0.32 mg/L.

 

Description of key information

48-hour EL*50 = 43 mg/L loading rate WAF; 48-hour NOEC =  32 mg/L loading rate WAF, OECD 202, EU Method C.2, Priestly & Mullee (2010)

Key value for chemical safety assessment

Additional information

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours under at a temperature of 21°C under static test conditions. The number of immobilised Daphnia 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.

Under the conditions of the test, the48-hour EL*50 for the test material, based on nominal loading rates, was 43 mg/L loading rate WAF with 95% confidence limits of 41 - 46 mg/L loading rate WAF. The No Observed Effect Loading rate was 32 mg/L loading rate WAF.

The 48-hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.78 mg/L with 95% confidence limits of 0.58 – 0.72 mg/L. The No Observed Effect Concentration was 0.32 mg/L.