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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-25 to 2010-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
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 (incl. QA statement)
Specific details on test material used for the study:
- Name as cited in study report: POPDA
- Color: colorless
- Lot/Batch number: LL100265
- Date receipt: 2010-09-21
- Expiry date: 2010-12-31
- Storage: in the dark
Analytical monitoring:
yes
Details on sampling:
- Range-finding:
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.

- Definitive:
Water samples were taken from the control and each test groups (replicates R1 , R2 pooled) at 0 and 48 hours for quantitative analysis. The 0-Hour 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:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
-Range-finding test
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 0.010, 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. Aliquots (100, 180, 320 and 560 ml) of the 100 mg/l test concentration were each separately diluted in a final volume of 1 litre of reconstituted water to give the 10, 18, 32 and 56 mg/l test concentrations respectively.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Positive control:
A positive control (Harlan Laboratories Ltd Project No: 0039/1160) 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.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.


Test organisms (species):
Daphnia magna
Details on test organisms:
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:
no
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 20ºC throughout the test.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
See Appendix 4 for results.
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 reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See Appendix 4 for results.
Salinity:
Not applicable (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 the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20ºC 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. 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.

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 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20ºC 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.

-Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a WTW pH Oxi 340I pH and dissolved oxygen meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL of 67 - 100 mg/l.
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
18 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:
-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 (see any other information on results).
No immobilisation was observed at the test concentrations of 0.010, 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/l test preparations at 0 and 48 hours showed measured test concentrations to range from 86% and 90% of nominal. These results indicated that the test item was stable in the test medium over the test period and under frozen storage conditions.
-Definitive Test
-Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2(see any other information on results). The relationship between percentage immobilisation and concentration at 48 hours is given in attached Figure 1.
Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits(mg/l)
24 >100 -
48 80 67 - 100

The No Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 18 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and its standard error of the response curve at 48 hours was 5.4 (SE = 1.3).

-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 3(see any other information on results). Temperature was maintained at approximately 20ºC throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed 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 2) showed measured test concentrations to range from 100% to 110% of nominal value and so it was considered justifiable to calculate 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 2 and 3.
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 were 8.5
(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 EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.19).

Reported statistics and error estimates:
An estimate of the EC50 value at 24 hours was given by inspection of the immobilisation data.
The EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.

-Evaluation of data for the positive control
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curves and standard errors were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used when two or more partial responses to exposure are shown.

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

0.010

0

0

0.10

0

0

1.0

0

0

10

0

0

100

3

9

 

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(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

0

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

0

32

0

0

0

0

0

1

1

5

56

0

0

0

0

2

0

2

10

100

2

1

3

15

8

7

15

75

 

R1¿ R2= Replicates 1 and 2

Table3              Cumulative Immobilisation Data in the Positive Control

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(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

Appendix3      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

9.1

100

20

21

8.0

8.8

99

21

 

R2

7.9

9.0

99

20

21

8.0

8.7

98

21

10

R1

8.7

9.2

101

20

21

8.2

8.7

98

21

 

R2

8.7

9.2

101

20

21

8.2

8.7

98

21

18

R1

9.0

9.4

103

20

21

8.3

8.7

98

21

 

R2

9.0

9.4

103

20

21

8.3

8.7

98

21

32

R1

9.2

9.5

104

20

21

8.6

8.7

98

21

 

R2

9.2

9.6

105

20

21

8.6

8.7

98

21

56

R1

9.4

9.8

108

20

21

8.6

8.7

98

21

 

R2

9.4

9.7

107

20

21

8.7

8.7

98

21

100

R1

9.7

9.8

108

20

21

8.9

8.8

99

21

 

R2

9.7

9.8

108

20

21

8.8

8.7

98

21

 


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

R1- R2= Replicates 1 and 2

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 value of 80 mg/l with 95% confidence limits of 67 - 100 mg/l. The No Observed Effect Concentration at 48 hours was 18 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 (2 replicates of 10 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 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.

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 understatic 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 to Daphnia magna based on nominal test concentrations was 80 mg/l with 95% confidence limits of 67 - 100 mg/l. The No Observed Effect Concentration was 18 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 100% to 110% of nominal value and so the results are based on nominal test concentrations only.

The 48-Hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.75 mg/l with 95% confidence limits of 0.65 - 0.86 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-07-28 to 2005-11-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Conducted according to ISO TC147/SC5/WG2 Guidelines. No information given on test substance purity or stability (responsibility of the Sponsor). No analytical monitoring of the test concentrations.
Qualifier:
according to guideline
Guideline:
other: ISO TC147/SC5/WG2
Deviations:
yes
Remarks:
No data on purity or stability of test substance.
GLP compliance:
yes
Remarks:
Statement-no certificate
Specific details on test material used for the study:
- Name as cited in study report: Jeffamine D-230
- color: colorless
- substance type: Active
- Lot/Batch number: 4W512
- Date receipt: 2005-07-05 727.9g; 2005-07-13 750.9g
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dissolution of test substance directly into control water
- Eluate: Synthetic seawater (Hawiian Marine Mix in deionized water, 25 +/- 1 ppt salinity)
Test organisms (species):
other aquatic crustacea: Acartia tonsa
Details on test organisms:
TEST ORGANISM
- Common name: Marine copepods
- Source: Stillmeadow laboratory culture
- Age at study initiation (mean and range, SD): 19-25 days
- Feeding during test
- Amount: Not fed during test

Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Test temperature:
Initial range-21 °C
Final range-20 °C
pH:
Initial range-8.2-9.8 SU
Final range-7.9-8.3
Dissolved oxygen:
Initial range-7.2-7.5 mg/L
Final range-6.8-7.0 mg/L
Salinity:
Initial-26 ppt
Fina lrange-26-27 ppt
Nominal and measured concentrations:
Nominal (Definitive test )-0, 25, 50, 100, 200, 400 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: plastic cups
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: polypropylene, 30 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): static
- 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: Hawaiian Marine Mix in deionized water, to 25 +/- 1 ppt
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, 24 and 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 0.1, 1.0, 10, 100, 500, 1000 mg/L (for range-finding)
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (50 mg/L)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
418.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL-279.07-3777.02 mg/L.
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality of control: 5 % (negative control)
Results with reference substance (positive control):
- Results with reference substance valid? Validity criteria not reported.
- Mortality: Test 1-20/20 organisms dead at 24 h. Test 2-15/20 organisms dead at 24 h; 20/20 dead at 48 h.
- EC50/LC50: Could not be determined.
Reported statistics and error estimates:
The EC50 was determined using the Probit linear regression model.
Validity criteria fulfilled:
yes
Remarks:
Control mortality <10%
Conclusions:
The 48 hr EC50 of the test substance to the marine copepod, Acartia tonsa, was determined to be 418.34 mg/L, with 95% confidence limits of 279.07-3777.02 mg/L. The 48 hr NOEC was determined to be 200 mg/L.

Description of key information

The 48-Hour EC50 of the test substance to Daphnia magna in freshwater was found to be 80 mg/L, with 95% confidence limits of 67 -100 mg/L. The No Observed Effect Concentration (NOEC) was 18 mg/L (Goodband & Mullee, 2010). The 48 -h EC50 of the test substance to the marine copepod, Acartia tonsa, was determined to be 418.34 mg/L, with 95% confidence limits of 279-3777 mg/L. The 48-h NOEC was determined to be 200 mg/L (Stillmeadow Inc., 2005).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
80 mg/L

Marine water invertebrates

Marine water invertebrates
Effect concentration:
418.34 mg/L

Additional information

A static, 48-h acute study, following GLP and ISO TC147/SC5/WG2 Guidelines was conducted with the marine copepod Acartia tonsa. The organisms used were 19 -25 days old, from an in-house culture. The salinity of the synthetic media was 25 -27 ppt. Concentrations used were Control, 25, 50, 100, 200, 400 mg/L, and 4 replicates of 5 animals each were exposed per concentration. Test temperature was 20 -21 °C. The 48 hr. EC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to the marine copepod, Acartia tonsa, was determined to be 418.34 mg/L, with 95% confidence limits of 279-3777 mg/L. The 48 -h NOEC was determined to be 200 mg/L.

Another 48 h static acute study was conducted with the freshwater crustacean, Daphnia magna. This test followed GLP and OECD Guideline 202. Neonates from an in-house culture were used, and were <24 hr old at test initiation. Two replicates of 10 organisms each were exposed to Control and 5 concentrations up to and including 100 mg/L. The 48-Hour EC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Daphnia magna was found to be 80 mg/L, with 95% confidence limits of 67 - 100 mg/L. The No Observed Effect Concentration (NOEC) was 18 mg/L.