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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 May 2018 and 16 June 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
These changes were considered to have had no impact on the outcome of the study as the overall conduct of the study remains the same.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: PG-RAW-0004
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 ºC in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the freshly prepared control and each test group from the bulk test preparation at 0 and 24 hours and from the old test media (pooled replicates) at 24 and 48 hours for quantitative analysis. Samples were taken for immediate analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 10 mg/L could be obtained using a saturated solution method of preparation.
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 0.10, 1.0, 10 and 100% v/v saturated solution.
A nominal amount of test item (58 mg) was dispersed in 580 mL of test water and shaken in an INFORS Multitron Incubator set at 200 rpm for 24 hours at 21 °C. After 24 hours the shaking was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL discarded in order to pre condition the filter), to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Initial Experiment
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% v/v saturated solution.

Definitive Test
The 100% v/v saturated solution was prepared as previously described, however, in the range finding test the initial stock solution was shaken at a temperature of 21 °C whereas the stock solution for the definitive test and initial experiment was shaken at 24°C. This was considered to have had no adverse effects on the outcome of the study. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution.
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 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 GEMMA Micro 300 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
18 to 22 °C with a maximum deviation of ±1 °C
pH:
pH 7.8-8.2
Dissolved oxygen:
7.9 - 9.2 mg O2/L
Nominal and measured concentrations:
Range-finding Test:
In the range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.054 to 7.9 mg/L. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions, and therefore a semi-static daily renewal approach was adopted for the definitive test.

Definitive Test:
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% v/v saturated solution.
Geometric Mean Measured Test Concentration (mg/L): 0.96, 2.1, 3.8, 6.9, 13

Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 1.0 to 14 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.80 and 11 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.
Details on test conditions:
Test Water
Reconstituted water (Elendt M7 medium) used for the range-finding test, initial experiment and definitive test.

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ±0.3 and stored at approximately 21 ºC

Range-finding Test
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range finding test five 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 shielded from laboratory light for a period of 48 hours. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 150 mL of test media and was sealed due to the volatile nature of the test item. 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.
Due to the potentially unstable nature of the test item in the light, all preparation and testing was conducted under laboratory safety lighting/shielded from the light.
Due to the potential volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were taken for immediate analysis.

Initial Experiment
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% v/v saturated solution.
An initial experiment conducted showed more than 10% of the control group daphnids were immobilized, therefore the test validity criteria was not met.
The results from the initial experiment were not used for reporting purposes.

Definitive Test
The 100% v/v saturated solution was prepared as previously described, however, in the range finding test the initial stock solution was shaken at a temperature of 21 °C whereas the stock solution for the definitive test and initial experiment was shaken at 24°C. This was considered to have had no adverse effects on the outcome of the study. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution.
The concentration and stability of the test item in the fresh test preparations was verified by chemical analysis at 0 and 24 hours from the bulk preparations. Analysis of the aged test preparations was verified by chemical analysis at 24 and 48 hours from the pooled replicates 1 to 4.

Exposure Conditions
In the definitive test 150 mL conical flasks 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 sealed 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.
Semi static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 Hour old test media into the fresh test media. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.

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
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal and on termination of the test after 48 hours represent those of the used or 24 hour old test preparations. 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 appearance of the test media was recorded daily.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL 2.1 – 3.8 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL 2.1 – 3.8 mg/L
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution, however, 10% immobilization was observed at 10% v/v saturated solution and 100% immobilization was observed at 100% v/v saturated solution.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.054 to 7.9 mg/L. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions, and therefore a semi-static daily renewal approach was adopted for the definitive test.

Definitive Test
Immobilization Data
Analysis of the immobilization data using the binomial method at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:
Time (hour) EC50 (mg/L) 95% Confidence limits(mg/L)
24 2.8 2.1 – 3.8
48 2.8 2.1 – 3.8
The NOEC based on immobilization after 24 and 48 hours exposure was 2.1 mg/L. The LOEC based on immobilization after 24 and 48 hours exposure was 3.8 mg/L.
Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 and 48 hours.

Sub-Lethal Effects
Reduced mobility was observed in the 2.1 mg/L test concentration.

Validation Criteria
The test was considered to be valid given that none 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
Temperature was maintained at 20 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Results with reference substance (positive control):
A positive control (Envigo study number LK67NP) 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 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) NOEC (mg/L) LOEC(mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

The NOEC 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*.

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration
(% v/v saturated solution)

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

5N

5 N

0

0

5 N

5 N

0.10

0

0

5 N

5 N

0

0

5 N

5 N

1.0

0

0

5 N

5 N

0

0

5 N

5 N

10

0

0

5 N

5 N

0

1

5 N

4 N

100

5

5

AI

AI

5

5

AI

AI

R      = Replicate

N      = Normal

AI     = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Concentration (mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

0.96

0

0

0

0

0

0

5 N

5 N

5 N

5 N

2.1

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.8

5

5

5

5

20

100

AI

AI

AI

AI

6.9

5

5

5

5

20

100

AI

AI

AI

AI

13

5

5

5

5

20

100

AI

AI

AI

AI

 

Geometric Mean Measured Concentration (mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.96

0

0

0

0

0

0

5 N

5 N

5 N

5 N

2.1

0

0

0

0

0

0

2R3 N

5 N

2 R 3 N

1 R 4 N

3.8

5

5

5

5

20

100

AI

AI

AI

AI

6.9

5

5

5

5

20

100

AI

AI

AI

AI

13

5

5

5

5

20

100

AI

AI

AI

AI

Rep   = Replicate

N      = Normal

R      = Reduced mobility

AI     = All daphnia immobilized

Water Quality Measurements

Geometric Mean Measured Concentration (mg/L)

0 Hours
(Fresh Media)

24 Hours
(Old Media)

pH

mg O2/L

Temperature
°C

pH

mg O2/L

Temperature
°C

Control

R1

7.9

9.2

20

8.0

8.7

21

0.96

R1

7.9

9.1

21

7.9

8.5

21

2.1

R1

8.0

9.0

21

7.9

8.3

21

3.8

R1

8.0

9.0

21

7.8

7.9

21

6.9

R1

8.0

8.8

21

7.8

7.8

21

13

R1

7.9

8.6

21

7.9

8.2

21

 

Geometric Mean Measured Concentration (mg/L)

24 Hours
(Fresh Media)

48 Hours
(Old Media)

pH

mg O2/L

Temperature
°C

pH

mg O2/L

Temperature
°C

Control

R1

8.2

8.9

20

7.9

8.6

20

0.96

R1

8.1

8.9

21

7.8

8.3

21

2.1

R1

8.1

8.8

21

7.9

8.4

21

3.8

R1

-

-

-

-

-

-

6.9

R1

-

-

-

-

-

-

13

R1

-

-

-

-

-

-

R= Replicate

-= No data, all daphnia immobilized

Validity criteria fulfilled:
yes
Conclusions:
The 48 h EC50 (immobilisation) value with Daphnia magna is 2.8 mg/I.
Executive summary:

A study was performed to assess the acute toxicity of PG-RAW-0004 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 10 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range‑finding test and initial experiment, 20 daphnids (four replicates of five animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under semi-static test conditions. The test item solutions were prepared by shaking an excess (100 mg/L) of test item in an INFORS Multitron Incubator set at 200 rpm for 24 hours. After the shaking period any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL discarded in order to pre-condition the filter), to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Due to the potentially unstable nature of the test item in the light, preparation was conducted under laboratory safety lighting/shielded from the light.

Due to the potential volatile nature of the test item, testing was conducted in stoppered test vessels in order to minimize possible losses due to volatilization. 

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 1.0 to 14 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.80 and 11 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.96, 2.1, 3.8, 6.9 and 13 mg/L.

Exposure of Daphnia magna to the test item gave a 48 h EC50 of 2.8 mg/L based on the geometric mean measured test concentrations.

Description of key information

A study was performed to assess the acute toxicity of PG-RAW-0004 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 10 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range‑finding test and initial experiment, 20 daphnids (four replicates of five animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under semi-static test conditions. The test item solutions were prepared by shaking an excess (100 mg/L) of test item in an INFORS Multitron Incubator set at 200 rpm for 24 hours. After the shaking period any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL discarded in order to pre‑condition the filter), to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Due to the potentially unstable nature of the test item in the light, preparation was conducted under laboratory safety lighting/shielded from the light.

Due to the potential volatile nature of the test item, testing was conducted in stoppered test vessels in order to minimize possible losses due to volatilization. 

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 1.0 to 14 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.80 and 11 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.96, 2.1, 3.8, 6.9 and 13 mg/L.

Exposure of Daphnia magna to the test item gave a 48 h EC50of 2.8 mg/L based on the geometric mean measured test concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.8 mg/L

Additional information