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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:
The study was conducted between 16 January 2012 and 29 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19-21 July 2011; Date of signature: 31 Aug 2011
Specific details on test material used for the study:
Sponsor's identification: Lemonile
Description: clear colourless liquid
Purity: 99.9%
Batch number: PE00027352
Date received:03 October 2011
Expiry date: 26 October 2013
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control (replicates R1 – R4 pooled) and each test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Information provided by the Sponsor indicated the water solubility of the test item to be 45 mg/l. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 41 mg/l was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

An amount of test item (2250 mg) was dispersed in 22.5 litres of Elendt M7 medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 41 mg/l.

A series of dilutions was made from this saturated solution to give the required nominal test concentrations of 4.1, 2.3, 1.3, 0.74 and 0.41 mg/l.
The stock solution and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Due to the light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting and/or shielded from the light
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Source: Derived from in-house laboratory cultures
- Age at study initiation:< 24 h
- Method of breeding: Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium (see Appendix 2) in a temperature controlled room at approximately 20C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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: No

Test type:
static
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperature was maintained at approximately 20°C throughout the test. Refer to table 3 in results section for temperature measurements.
pH:
Refer to table 3 in results section for pH measurements
Nominal and measured concentrations:
0.41, 0.74, 1.3, 2.3 and 4.1 mg/l. (nominal test concentrations)
0.501, 0.771, 1.36, 2.40 and 4.30 mg/l (measured test concentrations at 24 hours)
0.352, 0.597, 1.03, 2.27 and 4.04 mg/l (measured test concentrations at 48 hours)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml ground glass stoppered conical flasks completely filled with test preparation. The test vessels were then sealed to reduce losses due to volatilisation
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Elendt M7 medium was used. The theoretical total water hardness of Elendt M7 medium is 250 mg/l as CaCO3 therefore, in order to comply with the water characteristics for the OPPTS guideline, the prepared Elendt M7 was diluted in a ratio of 50:50 with deionised reverse osmosis water prior to use to reduce the total water hardness


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
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.041, 0.41, 4.1 and 41 mg/l.
Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 0.41, 0.74, 1.3, 2.3 and 4.1 mg/l.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL = 2.7 - 3.4
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL = 2.4- 3.0
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range finding test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilisation was observed at the test concentrations of 0.041 and 0.41 mg/l. However, immobilisation was observed at 4.1 and 41 mg/l.
Based on this information test concentrations of 0.41, 0.74, 1.3, 2.3 and 4.1 mg/l were selected for the definitive test.
Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed near nominal test concentrations were obtained indicating that the test item was stable under test conditions.

Definitive test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. 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 trimmed Spearman-Karber method (Hamilton et al 1977) at 24 and 48 hours based on the mean measured test concentrations are shown above.

The No Observed Effect Concentration after 24 and 48 hours exposure was 1.2 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration

Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 24 and 48 hours.

Observations on test item solubility:
All control and 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 6. Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The water temperature was also recorded in one control vessel every hour using a digital temperature logger. The temperature logger recorded temperatures of 20°C.

The oxygen concentration in some of the test vessels at 0 hours 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.

Verification of test concentrations:
The test item was known to be volatile (Henry’s law constant 21 Pa.m3/mol) and hence it was likely that losses of test item would occur during the time taken to prepare the required test concentrations. As such, samples were taken for immediate chemical analysis to minimise losses as far as practically possible. Chemical analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal with the exception of the 0.41 mg/l test preparation which showed a measured concentration of 122% of nominal was obtained. Analysis of the test preparations at 48 hours showed measured test concentrations to be near nominal with the exception of the 1.3 mg/l test preparation which showed a measured concentration of 79% of nominal was obtained. Given that the average measured concentrations were within 80-120% of nominal it was considered appropriate to base the results on the mean measured test concentrations which were determined to be 0.43, 0.68, 1.2, 2.3 and 4.2 mg/l
Results with reference substance (positive control):
Analysis of the immobilisation data by the geometric mean method at 24 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 95% CL
24 1.3 1.0 - 1.8
48 1.1 1.0 - 1.3
The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.56 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 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

Table1 Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.041

0

0

0.41

0

0

4.1

1

10

41

10

10

 

Table 2  Cumulative Immobilisation Data in the Definitive Test

Mean Measured Test Concentration

(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

0.43

0

0

0

0

0

0

0

0

0

0

0

0

0.68

0

0

0

0

0

0

0

0

0

0

0

0

1.2

0

0

0

0

0

0

0

0

0

0

0

0

2.3

1

1

0

0

2

10

1

1

2

1

5

25

4.2

5

4

5

5

19

95

5

5

5

5

20

100

R1– R2= Replicates 1 and 2

Table 3: Physico-Chemical Measurements

Mean Measured Test Concentration
(mg/l)

0 Hours

24 Hours

48 Hours

pH

mg O2/l

%*

T°C

TºC

pH

mg O2/l

%*

T°C

Control

R1

8.0

9.4

103

20

20

7.6

7.9

87

20

R2

8.0

9.3

102

20

20

7.9

8.8

97

20

R3

8.0

9.3

102

20

20

8.0

9.0

99

20

R4

8.0

9.3

102

20

20

7.9

8.8

99

21

0.43

 

R1

8.1

9.3

102

20

20

7.9

8.9

100

21

R2

8.2

9.3

102

20

20

7.9

8.7

98

21

R3

8.2

9.2

101

20

20

7.9

8.8

99

21

R4

8.1

9.2

101

20

20

7.9

8.7

98

21

0.68

R1

8.1

9.2

101

20

20

7.9

8.9

98

20

R2

8.1

9.2

101

20

20

8.0

8.9

100

21

R3

8.1

9.2

101

20

20

8.0

8.7

98

21

R4

8.1

9.2

101

20

20

8.0

8.7

98

21

1.2

R1

8.1

9.2

101

20

20

8.0

8.8

97

20

R2

8.1

9.2

101

20

20

8.1

8.7

98

21

R3

8.1

9.2

101

20

20

8.0

8.8

99

21

R4

8.1

9.2

101

20

20

8.0

8.8

99

21

2.3

R1

8.1

9.2

101

20

20

8.0

8.8

97

20

R2

8.1

9.2

101

20

20

8.0

8.8

97

20

R3

8.1

9.1

100

20

20

8.1

8.8

97

20

R4

8.1

9.1

100

20

20

8.0

8.8

97

20

4.2

R1

8.0

9.2

101

20

20

8.0

8.8

97

20

R2

8.1

9.1

100

20

20

8.0

8.8

97

20

R3

8.1

9.1

100

20

20

8.0

8.8

97

20

R4

8.1

9.1

100

20

20

8.1

8.8

97

20


*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1– R4= Replicates 1 to 4

Conclusions:
The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 2.7 mg/l with 95% confidence limits of 2.4 – 3.0 mg/l. The No Observed Effect Concentration was 1.2 mg/l.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the mean measured test concentrations gave a 48-Hour EC50 value of 2.7 mg/l with 95% confidence limits of 2.4 – 3.0 mg/l.  The No Observed Effect Concentration at 48 hours was 1.2 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.7 mg/L

Additional information

The key study was performed according to GLP and OECD Guideline 202. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at mean measured concentrations of 0.43, 0.68, 1.2, 2.3 and 4.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 2.7 mg/l with 95% confidence limits of 2.4 – 3.0 mg/l. The No Observed Effect Concentration was 1.2 mg/l.