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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 07 August 2017 and 24 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EDA-BADGE-EDA
Cas Number:
854009-15-7
Molecular formula:
C25H40N4O4
IUPAC Name:
EDA-BADGE-EDA
Constituent 2
Chemical structure
Reference substance name:
EDA-BADGE-EDA-BADGE-EDA
Molecular formula:
C48H72N6O8
IUPAC Name:
EDA-BADGE-EDA-BADGE-EDA
Constituent 3
Chemical structure
Reference substance name:
EDA-BADGE α-glycol
Molecular formula:
C23H34N2O5
IUPAC Name:
EDA-BADGE α-glycol
Constituent 4
Reference substance name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Constituent 5
Reference substance name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Test material form:
liquid: viscous
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
Physical state/Appearance: Yellow/brown extremely viscous liquid
Batch: BBF01102V1
Purity: Not provided
Expiry Date: 01 January 2021
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each loading rate WAF for TOC analysis at 0 and 48 hours

Test solutions

Vehicle:
yes
Remarks:
Reconstituted water (Elendt M7medium)
Details on test solutions:
Validation of Mixing Period
Preliminary work (see "Details on TEst Conditions", below) was carried out to determine whether stirring for a prolonged period produced significantly higher levels of Total Organic Carbon (TOC), as an indicator of soluble organic substances, in the WAF.


Range-finding Test
The loading rates 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 loading rates of 1.0, 10 and 100 mg/L
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. Prior to use, the test item was heated at 50 °C to aid weighing. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first
75-100 mL discarded) to give the 1.0 and 10 mg/L loading rate WAFs. Microscopic observations of the 1.0, 10 and 100 mg/L WAFs were performed after filtering and showed that the filtration had removed the undissolved particles in the 1.0 and 10 mg/L loading rates however, undissolved material was still present in the 100 mg/L loading rate. As a result, the 100 mg/L loading rate was further filtered twice through filter paper. After filtration through the filter paper, the 100 mg/L loading rate was still observed to be a slightly cloudy dispersion, however, it was considered that further filtration would be unsuccessful at removing any further dispersed material


Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Nominal amounts of test item (5.0, 9.0, 16, 28 and 50 mg) were each separately added to a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. Prior to use, the test item was heated at 50 °C in order to aid weighing. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column of the 10 mg/L loading rate and hence it was considered justifiable to remove the 10 mg/L loading rate WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed that no undissolved material remained. The 1.0, 1.8, 3.2 and 5.6 mg/L loading rates were removed by siphon without filtration through glass wool.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 1) in a temperature controlled room maintaining the water temperature at 18 to 22 °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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
22”C
pH:
The pH during the test was 7.9 - 8.2
Dissolved oxygen:
The dissolved oxygen concentration was 8.0 - 8.7 mg O2/L
Salinity:
Not reported
Conductivity:
Not reported
Details on test conditions:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of Total Organic Carbon (TOC), as an indicator of soluble organic substances, in the WAF.

Validation of Mixing Period
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of Total Organic Carbon (TOC), as an indicator of soluble organic items, in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by filtration through glass wool and filter paper and samples taken for TOC analysis
The results are summarized as follows:
Nominal Loading Rate(mg/L) Time (Hours)
24 96
mg C/L mg C/L
100 30.59 53.18

Whilst there was a significant increase in the measured carbon concentration obtained when the stirring period was extended beyond 24-Hours, visual observations made on the 96-Hour preparation after filtration showed that dispersed test item remained which may have caused physiological effects on the test organisms. As a result, the preparation of the WAF was maintained at 24 hours.

Range-finding Test
The loading rates 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 loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. Prior to use, the test item was heated at 50 °C to aid weighing. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 and 10 mg/L loading rate WAFs. Microscopic observations of the 1.0, 10 and 100 mg/L WAFs were performed after filtering and showed that the filtration had removed the undissolved particles in the 1.0 and 10 mg/L loading rates however, undissolved material was still present in the 100 mg/L loading rate. As a result, the 100 mg/L loading rate was further filtered twice through filter paper. After filtration through the filter paper, the 100 mg/L loading rate was still observed to be a slightly cloudy dispersion, however, it was considered that further filtration would be unsuccessful at removing any further dispersed material.
In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C 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. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Nominal amounts of test item (5.0, 9.0, 16, 28 and 50 mg) were each separately added to a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. Prior to use, the test item was heated at 50 °C in order to aid weighing. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column of the 10 mg/L loading rate and hence it was considered justifiable to remove the 10 mg/L loading rate WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed that no undissolved material remained. The 1.0, 1.8, 3.2 and 5.6 mg/L loading rates were removed by siphon without filtration through glass wool.
Samples were taken from the control and each loading rate WAF for TOC analysis at 0 and 48 hours (see Table 1).

Exposure Conditions
As in the range-finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test five 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) 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.

Assessments
Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
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 Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Total Organic Carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each test group at 0 and 48 hours. Duplicate samples were taken and stored frozen for further analysis if necessary.

Data Evaluation

Statistical Analysis
The EL50 values at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 2.
No immobilization was observed at 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF.
A single daphnia in the control group was observed to be trapped at the surface after 24 hours exposure. Given that no more than 10% were affected, and the effect did not last to the end of the test, this was considered to be due to natural causes.
Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive test.


Definitive Test
Total Organic Carbon Analysis
Measured concentrations of between 1.5 and 5.7 mg C/L were obtained at 0 hours (see Table1) and between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 1.0 mg C/L, and 4.8 mg C/L at 48 hours.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 3.
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the following results:

Time (h) EL50 (mg/L Loading Rate WAF)
24 1.8
48 1.7

The No Observed Effect Loading rate after 24 and 48 hours exposure was 1.0 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 1.8 mg/L loading rate WAF.
The slopes and their standard errors of the response curves at 24 and 48 hours were 22 (SE = 0.12) and 23 (SE = 0.34) respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the control and the 1.8 mg/L loading rate test concentration. These responses were trapping at the surface and reduced movement (see Table 3).

Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at 22”C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 362 to 399 lux.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear colorless water columns with test item adhered to glass slide suspended in the water column. After 23 hours stirring and a 1-Hour standing period the 1.0, 1.8, 3.2 and 5.6 mg/L loading rates were observed to be clear colorless water columns with no test item left adhered to the glass slide whereas the 10 mg/L loading rate was observed to be a slightly hazy water column with no test item left adhered to the glass slide. Microscopic examination of the WAFs showed that microdispersions were observed to be present in the 10 mg/L loading rate water column and therefore it was considered justifiable to remove the 10 mg/L loading rate WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed material. During the test the control, 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear, colorless solutions

Results with reference substance (positive control):
A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0
The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Table 2: Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal
Loading Rate
(mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

R1

R2

R1

R2

R1

R2

R1

R2

Control

0

0

1 T 4 N

5 N

0

0

5 N

5 N

1.0

0

0

5 N

5 N

0

0

5 N

5 N

10

5

5

A/I

A/I

5

5

A/I

A/I

100

5

5

A/I

A/I

5

5

A/I

A/I

R1– R2= Replicates 1 to 2

N = No sub-lethal effects observed

T = Trapped at surface

A/I = All daphnia immobolized

Table 3: Cumulative Immobilization Data and Observations in the Definitive Test

24 Hours

Nominal
Loading Rate
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

4 N 1T

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

4

2

3

2

11

55

1 R

3 N

2 N

3 N

3.2

5

5

5

5

20

100

A/I

A/I

A/I

A/I

5.6

5

5

5

5

20

100

A/I

A/I

A/I

A/I

10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

48 Hours

Nominal
Loading Rate
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

4 N 1 T

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

4

2

5

2

13

65

1 R

3 N

A/I

3 N

3.2

5

5

5

5

20

100

A/I

A/I

A/I

A/I

5.6

5

5

5

5

20

100

A/I

A/I

A/I

A/I

10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

T = Trapped at surface

A/I = All daphnia immobilized

Table 4: Water Quality Measurements

Nominal
Loading Rate
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

T°C

T ºC

pH

mg O2/L

T°C

Control

R1

7.9

8.5

22

22

7.9

8.6

22

1.0

R1

8.0

8.5

22

22

7.9

8.8

22

1.8

R1

8.0

8.5

22

22

8.0

8.7

22

3.2

R1

8.0

8.5

22

22

8.0

8.7

22

5.6

R1

8.1

8.6

22

22

8.0

8.7

22

10

R1

8.2

8.6

22

22

8.0

8.7

22

R1= Replicate 1


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Considered to be valid - no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results at 48 Hours with EL50 (mg/L Loading Rate WAF):
No Observed Effect Loading Rate (NOEL) (mg/L) = 1.0
Lowest Observed Effect Loading Rate (LOEL) (mg/L) = 1.8
Executive summary:

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results…….

Samples of the control and each loading rate were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. Measured concentrations of between 1.5 and 5.7 mg C/L were obtained at 0 hours and between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 1.0 mg C/L, and 4.8 mg C/L at 48 hours.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure ofDaphnia magnato the test item gave the following results:

Time Point

(Hours)

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/L)

Lowest Observed Effect Loading Rate (LOEL) (mg/L)

48

1.7

Not determined due to nature of the data

1.0

1.8