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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:
2010-09-28 - 2010-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Sponsor's identification: Amine C8
Description: dark brown liquid
Batch number: T7-271109
Date received: 21 September 2010
Expiry date: 31 December 2011
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
-Range-finding Test
Samples of the 0.010, 0.10, 1.0, 10 and 100 mg/l test concentrations were taken at 0 and 48 hours and stored at approximately 20ºC to determine the stability of the test item in the test medium under frozen storage conditions.

- Sampling method:
Definitive Test
Water samples were taken from the control (replicates R1 ¿ R2 pooled) and each test group (replicates R1 - R2 pooled) at 0 and 48 hours (see attached Appendix 3). Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

- Sample storage conditions before analysis:
Samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
-Range-finding test
The test concentration used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l. The test item was dissolved directly in reconstituted water.
An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration from which serial dilutions were prepared in reconstituted water to give the remainder of the test series of 10, 1.0, 0.10 and 0.010 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

-Definitive Test
For the purpose of the definitive test the test item was dissolved directly in reconstituted water.
An amount of test item (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg/l test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Source/preparation of dilution water:

Appendix 1 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.

Test organisms (species):
Daphnia magna
Details on test organisms:
Test Species
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20ºC. 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 suspension of algae (Chlorella sp.). 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

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

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.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:

Temperature was maintained at 21ºC throughout the test.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 4 for results.
Dissolved oxygen:
The oxygen concentration in some of the control 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 significant adverse effects were observed in the control group.
See Appendix 4 for results.
Salinity:

freshwater used.
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l.

Based on the results of the range-finding test, a single test concentration of four replicates, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.
Details on test conditions:
TEST SYSTEM
- Test vessel:
-Range-finding test
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21ºC to 22ºC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Some of the temperatures were measured to be slightly in excess of the 20 ± 1ºC given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and the temperatures were within the test guideline specification.
Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

-Definitive test
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure 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.

-Observations on test item solubility
Throughout the duration of the test the control was observed to be a clear, colourless solution whilst the 100 mg/l test concentration was observed to be a very pale brown solution.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Details on results:

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EC50 (mg/l)
24 >100
48 >100
The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

-Observations on test item solubility
Throughout the duration of the test the control was observed to be a clear, colourless solution whilst the 100 mg/l test concentration was observed to be a very pale brown solution.

-Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at 21ºC throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and 100 mg/l test concentration throughout the test.
The oxygen concentration in some of the control 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 significant adverse effects were observed in the control group.

-Verification of test concentrations
Analysis of the test preparations at 0 and 48 hours (see attached Appendix 3) showed measured test concentrations to range from 86% to 89% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.
Results with reference substance (positive control):
- Results with reference substance valid?
Yes


-Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1160) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in attached Figures 1 and 2.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50(mg/l) 95% Confidence limits (mg/l)
24 1.3 1.1-1.5
48 0.75 0.65-0.86

The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slopes and standard error of the response curves at 24 and 48 hours were8.5 (SE = 1.8) and 8.4 (SE = 1.8) respectively. 
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50value calculated from all positive controls was 0.77 mg/l (sd = 0.19).
Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.
Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.010

0

0

0.10

0

0

1.0

0

0

10

0

0

100

0

0

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/l)

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

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0


R1¿ R4= Replicates 1 to 4

Table3              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

0

0

0

0

2

1

3

15

1.0

1

3

4

20

9

8

17

85

1.8

9

9

18

90

10

10

20

100

3.2

10

10

20

100

10

10

20

100

 

R1¿ R2= Replicates 1 and 2

Appendix 4      Physico-Chemical Measurements

Nominal
Concentration
(mg/l)

0 Hours

24 Hours

48 Hours

pH

mg O2/l

%*

T°C

TºC

pH

mg O2/l

%*

T°C

Control

R1

7.9

8.8

99

21

21

7.9

8.9

100

21

 

R2

7.9

8.8

99

21

21

7.9

9.4

106

21

 

R3

7.9

8.8

99

21

21

7.9

9.0

101

21

 

R4

7.9

8.8

99

21

21

7.9

9.0

101

21

100

 

R1

9.3

8.8

99

21

21

8.6

8.4

94

21

R2

9.3

8.8

99

21

21

8.6

8.4

94

21

R3

9.3

8.8

99

21

21

8.5

8.5

96

21

R4

9.3

8.8

99

21

21

8.6

8.5

96

21

 

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

R1¿ R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly, the No Observed Effect Concentration was 100 mg/l.
Executive summary:


Introduction.


A study was performed to assess the acute toxicity of the test item 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.


 


Methods.


Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/l for 48 hours at a temperature of 21°C under static test conditions. 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 was 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 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.


 


Results.


The 48-Hour EC50 for the test item toDaphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.


It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.


Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 86% to 89% of nominal value and so the results are based on nominal test concentrations only.


CONCLUSION


The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.


 



Description of key information

One study (Goodband & Mullee, 2010 - report  41003973) is considered as the key study for endpoint coverage. The study investigates the acute toxicity of the substance to Daphnia magna according to the OECD guideline 202, under static conditions in freshwater and with an exposure period of 48 hour. The 48h-EC50 was determined to be >100 mg/L . The study is given a Klimisch score of 1 and was conducted under GLP.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

The study of Goodband and Mullee  (2010 - report 41003973) was performed to assess the acute toxicity of the test item to Daphnia magna according to the 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 a concentration of 100 mg/l for 48 hours at a temperature of 21°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.


No immobilisation was observed during the 48-hour test period. The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 86% to 89% of nominal value and so the results are based on nominal test concentrations only.