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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:
27 February 2012 - 5 April 2012
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 (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R4 pooled) at 0 and 48 hours.
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 (especially for difficult test substances)
- Method: Water Accommodated Fractio (WAF); for details see "Any other information on materials and methods incl. tables".

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture
- Age at study initiation: 1st instar
- Method of breeding:
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 20 to 21 °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 ofthe study.
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.
Hardness:
250 mg/L as CaC03
Test temperature:
21-22 °C
pH:
8.0 ± 0.2
Dissolved oxygen:
97-102 %
Salinity:
not applicable
Nominal and measured concentrations:
Nominal loading: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jar
- Type (delete if not applicable): open
- Ffill volume: 200 ml
- Aeration: no
- 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: Reconstituted Water- Elendt M7 Medium
Solution Concentration of stock solution (mg/L)
(I) H3B03 57190
(II) MnCl2*4H20 7210
(III) LiCl 6120
(IV) RbCl 1420
(V) SrCl2*6H20 3040
(VI) NaBr 320
(VII) Na2Mo04*2H20 1260
(VIII) CuCl2*6H20 335
(IX) ZnCl2 260
(X) CoCl2*2H20 200
(XI) KI 65
(XII) Na2SeO3 43.8
(XIII) NH4V03 11.5
(XIV) Na2EDTA*2H20 5000
FeS04*7H20 1991
An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21 °C.

- Macro Nutrient Stock Solutions
Solution Concentration of stock solution (g/L)
(XV) CaCl2*2H20 293.80
(XVI) NaHC03 64.80
(XVII) MgS04*7H20 246.60
(XVIII) Na2Si03*9H20 50.00
(XIX)KCl 28.00
(XX)NaN03 2.74
(XXI) K2HP04 1.84
(XXII) KH2P04 1.43

- Vitamin Nutrients
Solution Concentration of stock solution (mg/L)
(XXIII) Thiamine hydrochloride 750
Cyanocobalamine (vitamin B 12) 10
D(+) biotin (vitamin H) 7.5

- The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and analiquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.

- Reconstituted water used for both the range-finding and definitive test was used:
i) Stock Solutions
CaCl2*2H20 11.76 g/L
MgS04*7H20 4.93 g/L
NaHC03 2.59 g/L
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. 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 CaC03.






OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: 675 - 689 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
A positive control 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.
Analysis of the immobilization 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:
- 24-h EC50 = 1.3 mg/L, 95% Confidence Iimits 1.0-1.8 mg/L
- 48-h EC50 = 1.1 mg/L, 95% Confidence Iimits 1.0-1.3 mg/L
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 immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

* HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethai Concentration in Toxicity Bioassays. Environ Sei Technol 11, 714-719.

Validation of Mixing Period

Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for Ionger than 24 hours.

Range-tinding Test

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. No immobilization was observed at 10 and 100 mg/L loading rate WAF. Based on this information, a single loading rate of four replicates, of 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Table 1: Cumulative Immobilization Data in the Range-tinding Test

Nominal Loading Rate (mg/L)

Cumulative Immobilized Daphnia

(Initial Population: 10 Per Replicate)

24 hours

48 hours

Control

0

0

10

0

0

100

0

0

Definitive Test

1. Immobilization Data

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

Table 2: Cumulative Immobilization Data in the Definitive Test

Nominal Loading Rate (mg/L)

Cumulative Immobilized 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

 

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Table 3: Results for daphnia immobilization

Time (h)

EL50 (mg/L)

95% Confidence limits

24

> 100

-

48

> 100

-

 

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. lt was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

2. Observations on Test ltem Solubility

Observations on the test media were carried out during the mixing and testing of the WAF. At the start of the mixing period the 100 mg/L loading rate was observed to have formed a clear colorless water column with flakes of test item floating at the surface. After 23 hours stirring the WAF was observed to have formed a clear colorless water column with flakes of test item floating at the surface and small particles dispersed throughout the water column. After a 1-Hour standing period the WAF was observed to have formed a clear colorless water column with flakes of test item floating at the surface and small particles dispersed throughout the water column and settled on the bottom of the mixing vessel. Visual inspection of the WAF showed particles of test item to be dispersed throughout the water column and therefore it was considered justifiable to remove the 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 test item. During the test the control and 100 mg/L loading rate were observed to be clear, colorless solutions.

3. Total Organic Carbon Analysis

Total Organic Carbon (TOC) analysis ofthe test preparations at 0 and 48 hours showed measured test concentrations of 1.63 and 1.62 mg C/L respectively were obtained.

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.

Validation of Mixing Period

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, 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 slight 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 siphon and samples taken for Total Organic Carbon analysis. The results are summarized as follows:

Table 4:

Nominal Loading Rate (mg/L)

Time (hours)

24 hours

96 hours

mg C/L

mg C/L corrected for control

mg C/L

mg C/L corrected for control

Control

<LOQ

-

<LOQ

-

100

11.41

11.41

9.23

9.23

 

It is evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Results of Total Organic Carbon Analysis

Table 5: Results of Total Organic Carbon Analysis 

Samples

Nominal Loading Rate (mg/L)

Concentration of TOC (mg C/L)

Concentration of TOC Corrected for Control (mg C/L)

0 Hours

(Fresh Media)

Control R1- R4

<LOQ

-

100 R1- R4

1.63

1.63

48 Hours

(Old Media)

Control R1- R4

<LOQ

-

100 R1- R4

1.62

1.62

 

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 EL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
This indicates that in the range of water solubility the test item was not toxic under the test conditions.
Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with 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.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. This indicates that in the range of water solubility the test item was not toxic under the test conditions. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF. Given that a suitable method of chemical analysis could not be developed to measure the dissolved test item concentration present in the WAF it was considered appropriate to take samples for Total Organic Carbon (TOC) analysis as an alternative. Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured test concentrations of 1.63 and 1.62 mg C/L respectively were obtained. 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.

*EL = Effective Loading Rate

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K1 study therefore K2 is the highest Klimisch score that can be assigned.
Justification for type of information:
Please see the read-across report attached in section 13 of this dossier.
Reason / purpose for cross-reference:
read-across: supporting information
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R4 pooled) at 0 and 48 hours.
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 (especially for difficult test substances)
- Method: Water Accommodated Fractio (WAF); for details see "Any other information on materials and methods incl. tables".

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture
- Age at study initiation: 1st instar
- Method of breeding:
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 20 to 21 °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 ofthe study.
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.
Hardness:
250 mg/L as CaC03
Test temperature:
21-22 °C
pH:
8.0 ± 0.2
Dissolved oxygen:
97-102 %
Salinity:
not applicable
Nominal and measured concentrations:
Nominal loading: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jar
- Type (delete if not applicable): open
- Ffill volume: 200 ml
- Aeration: no
- 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: Reconstituted Water- Elendt M7 Medium
Solution Concentration of stock solution (mg/L)
(I) H3B03 57190
(II) MnCl2*4H20 7210
(III) LiCl 6120
(IV) RbCl 1420
(V) SrCl2*6H20 3040
(VI) NaBr 320
(VII) Na2Mo04*2H20 1260
(VIII) CuCl2*6H20 335
(IX) ZnCl2 260
(X) CoCl2*2H20 200
(XI) KI 65
(XII) Na2SeO3 43.8
(XIII) NH4V03 11.5
(XIV) Na2EDTA*2H20 5000
FeS04*7H20 1991
An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21 °C.

- Macro Nutrient Stock Solutions
Solution Concentration of stock solution (g/L)
(XV) CaCl2*2H20 293.80
(XVI) NaHC03 64.80
(XVII) MgS04*7H20 246.60
(XVIII) Na2Si03*9H20 50.00
(XIX)KCl 28.00
(XX)NaN03 2.74
(XXI) K2HP04 1.84
(XXII) KH2P04 1.43

- Vitamin Nutrients
Solution Concentration of stock solution (mg/L)
(XXIII) Thiamine hydrochloride 750
Cyanocobalamine (vitamin B 12) 10
D(+) biotin (vitamin H) 7.5

- The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and analiquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.

- Reconstituted water used for both the range-finding and definitive test was used:
i) Stock Solutions
CaCl2*2H20 11.76 g/L
MgS04*7H20 4.93 g/L
NaHC03 2.59 g/L
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. 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 CaC03.






OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: 675 - 689 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
A positive control 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.
Analysis of the immobilization 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:
- 24-h EC50 = 1.3 mg/L, 95% Confidence Iimits 1.0-1.8 mg/L
- 48-h EC50 = 1.1 mg/L, 95% Confidence Iimits 1.0-1.3 mg/L
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 immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

* HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethai Concentration in Toxicity Bioassays. Environ Sei Technol 11, 714-719.

Validation of Mixing Period

Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for Ionger than 24 hours.

Range-finding Test

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. No immobilization was observed at 10 and 100 mg/L loading rate WAF. Based on this information, a single loading rate of four replicates, of 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

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

Nominal Loading Rate (mg/L)

Cumulative Immobilized Daphnia

(Initial Population: 10 Per Replicate)

24 hours

48 hours

Control

0

0

10

0

0

100

0

0

Definitive Test

1. Immobilization Data

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

Table 2: Cumulative Immobilization Data in the Definitive Test

Nominal Loading Rate (mg/L)

Cumulative Immobilized 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

 

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Table 3: Results for daphnia immobilization

Time (h)

EL50 (mg/L)

95% Confidence limits

24

> 100

-

48

> 100

-

 

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. lt was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

2. Observations on Test ltem Solubility

Observations on the test media were carried out during the mixing and testing of the WAF. At the start of the mixing period the 100 mg/L loading rate was observed to have formed a clear colorless water column with flakes of test item floating at the surface. After 23 hours stirring the WAF was observed to have formed a clear colorless water column with flakes of test item floating at the surface and small particles dispersed throughout the water column. After a 1-Hour standing period the WAF was observed to have formed a clear colorless water column with flakes of test item floating at the surface and small particles dispersed throughout the water column and settled on the bottom of the mixing vessel. Visual inspection of the WAF showed particles of test item to be dispersed throughout the water column and therefore it was considered justifiable to remove the 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 test item. During the test the control and 100 mg/L loading rate were observed to be clear, colorless solutions.

3. Total Organic Carbon Analysis

Total Organic Carbon (TOC) analysis ofthe test preparations at 0 and 48 hours showed measured test concentrations of 1.63 and 1.62 mg C/L respectively were obtained.

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.

Validation of Mixing Period

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, 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 slight 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 siphon and samples taken for Total Organic Carbon analysis. The results are summarized as follows:

Table 4:

Nominal Loading Rate (mg/L)

Time (hours)

24 hours

96 hours

mg C/L

mg C/L corrected for control

mg C/L

mg C/L corrected for control

Control

<LOQ

-

<LOQ

-

100

11.41

11.41

9.23

9.23

 

It is evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Results of Total Organic Carbon Analysis

Table 5: Results of Total Organic Carbon Analysis 

Samples

Nominal Loading Rate (mg/L)

Concentration of TOC (mg C/L)

Concentration of TOC Corrected for Control (mg C/L)

0 Hours

(Fresh Media)

Control R1- R4

<LOQ

-

100 R1- R4

1.63

1.63

48 Hours

(Old Media)

Control R1- R4

<LOQ

-

100 R1- R4

1.62

1.62

 

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 EL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
This indicates that in the range of water solubility the test item was not toxic under the test conditions.
Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with 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.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. This indicates that in the range of water solubility the test item was not toxic under the test conditions. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF. Given that a suitable method of chemical analysis could not be developed to measure the dissolved test item concentration present in the WAF it was considered appropriate to take samples for Total Organic Carbon (TOC) analysis as an alternative. Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured test concentrations of 1.63 and 1.62 mg C/L respectively were obtained. 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.

*EL = Effective Loading Rate

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. 
This indicates that in the range of water solubility the test item was not toxic under the test conditions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Marine water invertebrates

Marine water invertebrates
Effect concentration:
100 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with 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.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. This indicates that in the range of water solubility the test item was not toxic under the test conditions. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF. Given that a suitable method of chemical analysis could not be developed to measure the dissolved test item concentration present in the WAF it was considered appropriate to take samples for Total Organic Carbon (TOC) analysis as an alternative. Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured test concentrations of 1.63 and 1.62 mg C/L respectively were obtained. 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.

*EL = Effective Loading Rate