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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
March 27, 2018 to March 29, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
Analysis of the samples for the verification of exposure concentrations was performed using HPLC
Details on sampling:
Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.
Vehicle:
yes
Remarks:
Elendt M4 medium
Details on test solutions:
Stock solutions were prepared for both the range-finding and definitive tests by addition of the test sample directly to Elendt M4 medium in a volumetric flask of adequate volume. Calculated volumes of the stock solution were added to nutrient growth medium and made to volume to prepare the test concentrations and provide enough solution for testing and subsequent water quality measurements. The test concentrations for both the range-finding test and definitive test were prepared separately by the addition of the test sample to Elendt M4 medium in volumetric flasks of adequate volume to provide enough solution for testing and subsequent water quality measurements.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna
Source: From cultures maintained at Chemex Environmental International Ltd since July 2014 and originally sourced from a clone of genotype 5.
Date of gravid isolation: 26 March 2018
Age of Daphnia at start of test: ≤ 24 h
Culture conditions: Cultured under static conditions
Temperature: 20 ± 2°C
Dissolved oxygen: >60% (or 3mg/L) ASV
Photoperiod: 16 h light and 8 h dark
Feeding regime: Each working day, a suspension of Chlorella vulgaris. The Daphnia in the test were not fed.
The Daphnia were cultured, and the tests performed, in Elendt M4 media. The dissolved oxygen (DO) concentration was >60% air saturation and the pH stabilised between 6 and 9 before commencement of the test (see 2.5 Test methods and conditions).
Test type:
static
Water media type:
other: Elendt M4 media
Limit test:
no
Total exposure duration:
48 h
Test temperature:
19.9 – 20.7°C
pH:
6.64 – 7.72
Dissolved oxygen:
8.14 – 9.20 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 0.625, 1.25, 2.5, 5.0, and 10 mg/L
Mean measured: 0 (control), 0.585, 0.987, 1.786, 2.701 and 7.223 mg/L
Details on test conditions:
The test was carried out according to the procedures given below, and SOP E201 based on the OECD 202 guideline Daphnia sp. Acute Immobilisation Test.
Chemex SOP reference: E201 “Daphnia magna 24 & 48 hr EC50 acute immobilisation test”

(A) Preliminary test method
A preliminary (range-finding) test was conducted at concentrations of 0 (control), 0.1, 1, 10 and 100mg/L. The duration of the preliminary test was 48 ± 1 h. There was a single replicate at each concentration.
Preliminary test results: Data from the preliminary test identified the 48-h EC50 as being between 1-10mg/L.

(B) Definitive test method
Test period: 27 to 29 March 2018
Test duration: 48 ± 1 h
Test volume: 25 mL
Test vessel: 40 mL glass dishes. To limit aerial contamination of the test vessels and reduce evaporative losses, the vessels were covered with a perspex sheet.

Number of replicates: Four replicates at each concentration.

Test concentrations:
Nominal: 0 (control), 0.625, 1.25, 2.5, 5.0, and 10 mg/L
Mean measured: 0 (control), 0.585, 0.987, 1.786, 2.701 and 7.223 mg/L

Test media: Elendt M4 reconstituted Daphnia water
Test organism: Five Daphnia were transferred to each control and test concentration vessel using a wide bore glass tube in the minimum amount of culture water (<0.5 mL).
Replacement regime: Static

Test conditions: Initial pH at 0 H: 6.64 (Required: 6-9)
pH range in control and test concentrations throughout test: 6.64 – 7.72

Initial Dissolved Oxygen at 0 H: 8.91mg/L
DO range in control and test concentrations throughout test: 8.14 – 9.20mg/L (Required: ≥3mg/L)

Temperature range within incubator throughout test: 19.9 – 20.7 °C (Required: 20±2°C maintained to ±1°C)

Illumination: 16 H Light, 8 H Dark Cycle

Water quality measurements: The pH (to 0.01), dissolved oxygen mg/L (to 0.01) and temperature (to 0.1°C) measurements were taken at 0 h on remaining test solution, and at 48-h endpoint on pooled replicates for each test and control solution. Temperature of incubation was recorded at 0, 24 and 48 h.

Observation frequency: The number of immobilised Daphnia was recorded after 24 and 48-h exposure periods.

Analysis of test substance: Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

Calculation of results: Where possible, the EC(r)10, EC(r)50 EC(r)90 values with 95% confidence limits and NOEC(r) were calculated according to the statistical methods of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

Reference Substance: A separate GLP study (ENV 11395) was conducted from 18 to 20 January 2017 to monitor the sensitivity of the test animals and test procedures. The reference study gave a 24 h EC50 of 1.4mg/L of potassium dichromate which was within the acceptable range 0.6 mg/L to 2.1 mg/L as specified in Technical Corrigendum to ISO6341.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 2.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 2.13 - 5.27 mg/L
Remarks:
(LC50 value is equivalent to 1.53 mg a.i./L)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1.786 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Determined by Bonferroni t Test
Remarks:
(NOEC value is equivalent to 1.12 mga.i./L)
Results with reference substance (positive control):
A separate GLP study (ENV 11395) was conducted from 18 to 20 January 2017 to monitor the sensitivity of the test animals and test procedures. The reference study gave a 24 h EC50 of 1.4 mg/L of potassium dichromate which was within the acceptable range 0.6 mg/L to 2.1 mg/L as specified in Technical Corrigendum to ISO6341.

Results:

 

Cumulative immobilisation

Nominal Concentration

mg/L

Number immobilised

% immobilisation

24 h

48 h

24 h

48 h

0 (Control)

0

0

0

0

0.625

0

0

0

0

1.25

0

0

0

0

2.5

0

0

0

0

5.0

10

14

50

70

10

19

20

95

100

 

Observations

Nominal Concentration

mg/L

Recorded observations

24 h

48 h

0 (Control)

20 NS

20 NS

0.625

20 NS

20 NS

1.25

20 NS

20 NS

2.5

20 NS

20 NS

5.0

10 NS

6 NS

10

1 Ms D

-

 

Key to comments

S

Sedimentation

Ms

Movement Slower

D

Debris attached to Daphnia

TM

Trapped in Meniscus (mobile)

LI

Lighter Colouration

TI

Trapped in Meniscus (immobile)

NS

No Signs of Abnormality

SM

Daphnia Smaller

-

All Daphnia immobilised

 

EC50 values

Exposure period

(h)

EC50value

(mg/L)

95% confidence limits

(mg/L)

24

2.70 mg/L

2.05 - 5.87 mg/L

48

2.44 mg/L

2.13 - 5.27 mg/L

Statistical methods used in ToxCalc v5.0: Linear Interpolation

$Normality of distribution and equality of variances could not be confirmed.

All concentrations of the test substance are reported as mean measured concentrations after analytical confirmation.

Discussion

The definitive test conducted from 27 to 29 March 2018 was performed according to the OECD 202 2004 guideline and met all validity criteria. The 24-h EC50 and 48-h EC50 of Croquat WKP PE to Daphnia magna were 2.70mg/L and 2.44mg/L respectively (both determined by Linear Interpolation using mean measured concentrations). The 0 to 24-h NOEC and LOEC were 1.786mg/L and 2.701 mg/L respectively (determined by Bonferroni t Test using mean measured concentrations). The 0 to 48-h NOEC and LOEC were 1.786 mg/L and 2.701 mg/L respectively (determined by Bonferroni t Test using mean measured concentrations). The Daphnia were examined at 24-h and 48-h for abnormal behaviour during the determination of immobility. Abnormalities were observed during the test period, with 1 individual showing slower movement at 24 h at 10mg/L. The lowest test concentration which immobilised all twenty Daphnia after 48-h was 10 mg/L. The highest concentration where no immobilisation occurred was 2.5 mg/L. None (0%) of the twenty control Daphnia maintained in dilution water were immobilised and none were trapped in the meniscus during the study. This fulfills the control validity criteria of the study as the percentage of immobilisation did not exceed 10%, confirming that the test organisms were of suitable “health” for use in the study. The study fulfilled the water quality validity criteria. Specifically, the dissolved oxygen concentration remained greater than 3 mg/L (or 60%ASV) for the duration of the test.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the 48 h EC50 of the test substance for toxicity in Daphnia magna is considered to be 2.44 mg/L (measured), i.e., equivalent to 1.53 mg a.i./L
Executive summary:

A study was conducted to determine the acute toxicity of read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal read across substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h, under static conditions. The concentrations of read across substance in the test vessels were measured using the HPLC method. Based on the analysis, the mean measured concentrations of read across substance were determined to be 0, 0.585, 0.987, 1.786, 2.701 and 7.223 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Abnormalities were observed during the test period, with 1 individual showing slower movement at 24 h at 10 mg/L (nominal). The lowest test concentration which immobilised all twenty Daphnia after 48 h was 10 mg/L (nominal). The highest concentration where no immobilisation occurred was 2.5 mg/L (nominal or 1.786 mg/L measured). Based on immobility compared to the control (p <0.05), the 48 h NOEC, LOEC values were determined to be 1.786 and 2.701 mg/L (measured) respectively. The 48 h EC50 was calculated to be 2.44 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test had satisfied all validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for toxicity in Daphnia magna was determined to be 2.44 mg/L (measured), i.e., equivalent to 1.53 mg a.i./L (Chemex, 2018). Based on the results of the read across study, similar EC50 value is expected for the test substance, 'steardimonium hydroxypropyl hydrolysed wool' for toxicity to Daphnia.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
April 04, 2018 to April 06, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Deviation does not have effect on the results and had no impact on the integrity and validity of the study
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
Analysis of the samples for the verification of exposure concentrations was performed using HPLC
Details on sampling:
Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.
Vehicle:
yes
Remarks:
Elendt M4 medium
Details on test solutions:
Stock solutions were prepared for both the range-finding and definitive tests by addition of the test sample directly to Elendt M4 medium in a volumetric flask of adequate volume. Calculated volumes of the stock solution were added to nutrient growth medium and made to volume to prepare the test concentrations and provide enough solution for testing and subsequent water quality measurements. The test concentrations for both the range-finding test and definitive test were prepared separately by the addition of the test sample to Elendt M4 medium in volumetric flasks of adequate volume to provide enough solution for testing and subsequent water quality measurements.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna
Source: From cultures maintained at Chemex Environmental International Ltd since July 2014 and originally sourced from a clone of genotype 5.
Date of gravid isolation: 3 April 2018
Age of Daphnia at start of test: ≤ 24 h
Culture conditions: Cultured under static conditions
Temperature: 20 ± 2°C
Dissolved oxygen: >60% (or 3mg/L) ASV
Photoperiod: 16 h light and 8 h dark
Feeding regime: Each working day, a suspension of Chlorella vulgaris. The Daphnia in the test were not fed.
The Daphnia were cultured, and the tests performed, in Elendt M4 media. The dissolved oxygen (DO) concentration was >60% air saturation and the pH stabilised between 6 and 9 before commencement of the test.
Test type:
static
Water media type:
other: Elendt M4 media
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.0 - 21.3°C
pH:
6.81 - 7.78
Dissolved oxygen:
8.52 - 9.00 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 9.4, 17, 30, 55 and 100 mg/L
Mean measured: 0 (control), 4.738, 10.100, 19.396, 36.088 and 65.863 mg/L
Details on test conditions:
The test was carried out according to the procedures given below, and SOP E201 based on the OECD 202 guideline Daphnia sp. Acute Immobilisation Test.
Chemex SOP reference: E201 “Daphnia magna 24 & 48 hr EC50 acute immobilisation test”

(A) Preliminary test method
A preliminary (range-finding) test was conducted at concentrations of 0 (control), 0.1, 1, 10 and 100 mg/L. The duration of the preliminary test was 48 ± 1 h. There was a single replicate at each concentration.
Preliminary test results: Data from the preliminary test identified the 48-h EC50 as being between 10-100 mg/L.

(B) Definitive test method
Test period: 04 to 06 April 2018
Test duration: 48 ± 1 h
Test volume: 25 mL
Test vessel: 40 mL glass dishes. To limit aerial contamination of the test vessels and reduce evaporative losses, the vessels were covered with a perspex sheet.

Number of replicates: Four replicates at each concentration.

Test concentrations:
Nominal: 0 (control), 9.4, 17, 30, 55 and 100 mg/L
Mean measured: 0 (control), 4.738, 10.100, 19.396, 36.088 and 65.863 mg/L

Test media: Elendt M4 reconstituted Daphnia water
Test organism: Five Daphnia were transferred to each control and test concentration vessel using a wide bore glass tube in the minimum amount of culture water (<0.5 mL).
Replacement regime: Static

Test conditions:

Initial pH at 0 h: 7.73 (Required: 6 - 9)
pH range in control and test concentrations throughout test: 6.81 - 7.78

Initial Dissolved Oxygen at 0 h: 8.85 mg/L
DO range in control and test concentrations throughout test: 8.52 - 9.00 mg/L (Required: ≥3 mg/L)

Temperature range within incubator throughout test: 20.0 - 21.3°C (Required: 20 ± 2°C maintained to ± 1°C)

Illumination: 16 h Light, 8 h Dark Cycle

Water quality measurements: The pH (to 0.01), dissolved oxygen mg/L (to 0.01) and temperature (to 0.1°C) measurements were taken at 0 h on remaining test solution, and at 48-h endpoint on pooled replicates for each test and control solution. Temperature of incubation was recorded at 0, 24 and 48 h.

Observation frequency: The number of immobilised Daphnia was recorded after 24 and 48-h exposure periods.

Analysis of test substance: Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

Calculation of results: Where possible, the EC(r)10, EC(r)50, EC(r)90 values with 95% confidence limits and NOEC(r) were calculated according to the statistical methods of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

Reference Substance: A separate GLP study (ENV 11395) was conducted from 18 to 20 January 2017 to monitor the sensitivity of the test animals and test procedures. The reference study gave a 24 h EC50 of 1.4 mg/L of potassium dichromate which was within the acceptable range 0.6 mg/L to 2.1 mg/L as specified in Technical Corrigendum to ISO6341.

Study deviation: Definitive test states that the test will be performed in a temperature-controlled incubator at 20±2°C, varying by no more than ±1°C throughout. The temperature within the incubator had a recorded range between 20.0 – 21.3°C, 0.3°C outside the recommended ±1°C variation range. The temperature was still within the specified range of 20±2°C throughout the test. No mortality or abnormal behaviour was observed in the control animals thus any effects seen in the concentrations were due to the test material. Therefore a deviation of +0.3°C outside the specified variation range had no effect on the results and had no impact on the integrity and validity of the study.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 23.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 20.1 - 27.0 mg/L
Remarks:
(LC50 value is equivalent to 14.14 mg a.i./L)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: determined by Williams' Test
Remarks:
(NOEC is equivalent to 6.13 mg a.i./L)
Results with reference substance (positive control):
A separate GLP study (ENV 11395) was conducted from 18 to 20 January 2017 to monitor the sensitivity of the test animals and test procedures. The reference study gave a 24 h EC50 of 1.4mg/L of potassium dichromate which was within the acceptable range 0.6 mg/L to 2.1 mg/L as specified in Technical Corrigendum to ISO6341.

Results:

 

Cumulative immobilisation

Nominal Concentration

mg/L

Mean measured concentration mg/L

Number immobilised

% immobilisation

24 h

48 h

24 h

48 h

0 (Control)

0 control

0

0

0

0

9.4

4.738

0

0

0

0

17

10.100

0

0

0

0

30

19.396

2

5

10

25

55

36.088

14

19

70

95

100

65.863

20

20

100

100

Observations

Nominal Concentration

mg/L

Mean measured concentration

mg/L

Recorded observations

24 h

48 h

0 (Control)

0 control

20NS

20NS

9.4

4.738

20NS

20NS

17

10.100

20NS

20NS

30

19.396

18NS

15NS

55

36.088

3NS,3MS

1NS

100

65.863

-

-

 

Key to comments

S

Sedimentation

Ms

Movement Slower

D

Debris attached to Daphnia

TM

Trapped in Meniscus (mobile)

LI

Lighter Colouration

TI

Trapped in Meniscus (immobile)

NS

No Signs of Abnormality

SM

Daphnia Smaller

-

All Daphnia immobilised

 

EC50 value

Exposure period

(h)

EC50value

(mg/L)

95% confidence limits

(mg/L)

24

29.9

25.4 – 34.9

48

23.3

20.1 - 27.0

Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Probit. All concentrations of the test substance are reported as mean measured concentrations following analytical confirmation.

Discussion

The definitive test conducted from 04 to 06 April 2018 was performed according to the OECD 202 2004 guideline and met all validity criteria. The 24-h EC50 and 48-h EC50of test substance toDaphnia magnawere 29.9 mg/L and 23.3 mg/L respectively (determined byMaximum Likelihood-Probit). The 0 to 24-h NOEC and LOEC were 19.396 mg/L and 36.088 mg/L respectively (determined by Steel's Many-One Rank Test). The 0 to 48-h NOEC and LOEC were 10.100 mg/L and 19.396 mg/L respectively (determined by Williams' Test).TheDaphniawere examined at 24-h and 48-h for abnormalbehaviourduring the determination of immobility. Abnormalities were observed during the test period, with 3 individuals showing slower movement at 24 h in 36.088 mg/L mean measured test concentration. The lowest mean measured test concentration which immobilised all twentyDaphniaafter 48-h was 65.863 mg/L. The highest concentration where no immobilisation occurred was 10.100 mg/L. None (0%) of the twenty controlDaphniamaintained in dilution water were immobilised and none were trapped in the meniscus during the study. This fulfills the control validity criteria of the study as the percentage of immobilisation did not exceed 10%, confirming that the test organisms were of suitable “health” for use in the study.Analytical confirmation of the test concentrations was performed. The study fulfilled the water quality validity criteria. Specifically, the dissolved oxygen concentration remained greater than 3 mg/L (or 60% ASV) for the duration of the test.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the 48 h EC50 of the test substance for toxicity in Daphnia magna is considered to be 23.3 mg/L (measured), i.e., equivalent to 14.14 mg a.i./L.



Executive summary:

A study was conducted to determine the acute toxicity of read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: 60.7%), to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms each were exposed to nominal test substance concentrations of 0, 9.4, 17, 30, 55 and 100 mg/L for 48 h under static conditions. The concentrations of read across substance in the test vessels were measured using the HPLC method. The mean measured concentrations of read across substance were determined to be 0, 4.74, 10.1, 19.396, 36.09 and 65.86 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Abnormalities were observed during the test period, with 3 individuals showing slower movement at 24 h in 36.09 mg/L mean measured test concentration. The lowest mean measured test concentration which immobilised all twenty Daphnia after 48-h was 65.86 mg/L. The highest concentration where no immobilisation occurred was 10.1 mg/L. Based on immobility compared to the control (p <0.05), the 48 h NOEC was determined to be 10.1 mg/L (measured) and 48 h LOEC was determined to be 19.396 mg/L (measured). Based on the study results, the 48 h EC50 was calculated to be 23.3 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test had satisfied all the validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for toxicity in Daphnia magna was determined to be 23.3 mg/L (measured; i.e., equivalent to 14.14 mg a.i./L) (Chemex, 2018). Based on results of the read across study, similar EC50 value can be expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool' for toxicity to Daphnia.

Description of key information

Based on results of read across studies, the lower 48 h EC50 value of 1.53 mg a.i./L (measured) for toxicity to Daphnia, has been considered further for hazard assessment of the test substance, 'Steardimonium hydroxypropyl hydrolysed wool', as a conservative approach.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.53 mg/L

Additional information

In absence of acute toxicity study with the test substance in Daphnia magna, the endpoint has been assessed based on studies for read across substances. The results are presented below:

Study 1:

A study was conducted to determine the acute toxicity of read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal read across substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h, under static conditions. The concentrations of read across substance in the test vessels were measured using the HPLC method. Based on the analysis, the mean measured concentrations of read across substance were determined to be 0, 0.585, 0.987, 1.786, 2.701 and 7.223 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Abnormalities were observed during the test period, with 1 individual showing slower movement at 24 h at 10 mg/L (nominal). The lowest test concentration which immobilised all twenty Daphnia after 48 h was 10 mg/L (nominal). The highest concentration where no immobilisation occurred was 2.5 mg/L (nominal or 1.786 mg/L measured). Based on immobility compared to the control (p <0.05), the 48 h NOEC, LOEC values were determined to be 1.786 and 2.701 mg/L (measured) respectively. The 48 h EC50 was calculated to be 2.44 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test had satisfied all validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for toxicity in Daphnia magna was determined to be 2.44 mg/L (measured), i.e., equivalent to 1.53 mg a.i./L (Chemex, 2018).

Study 2:

A study was conducted to determine the acute toxicity of read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: 60.7%), to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms each were exposed to nominal test substance concentrations of 0, 9.4, 17, 30, 55 and 100 mg/L for 48 h under static conditions. The concentrations of read across substance in the test vessels were measured using the HPLC method. The mean measured concentrations of read across substance were determined to be 0, 4.74, 10.1, 19.396, 36.09 and 65.86 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Abnormalities were observed during the test period, with 3 individuals showing slower movement at 24 h in 36.09 mg/L mean measured test concentration. The lowest mean measured test concentration which immobilised all twenty Daphnia after 48-h was 65.86 mg/L. The highest concentration where no immobilisation occurred was 10.1 mg/L. Based on immobility compared to the control (p <0.05), the 48 h NOEC was determined to be 10.1 mg/L (measured) and 48 h LOEC was determined to be 19.396 mg/L (measured). Based on the study results, the 48 h EC50 was calculated to be 23.3 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test had satisfied all the validity criteria. Under the study conditions, the 48 h EC50 of the read across substance for toxicity in Daphnia magna was determined to be 23.3 mg/L (measured; i.e., equivalent to 14.14 mg a.i./L) (Chemex, 2018).

Further, the hydrolysed proteins differences across the read across substances is not expected to have an impact, as the hydrolysed proteins are considered to pose a low risk to aquatic organisms. They quickly degrade to more simple metabolites, called amino acids; hence their persistence in the environment is very short, without any tendency to bioaccumulation. Further, these compounds are present in live cells; consequently, they are not considered real waste, since they can be used again by the same live cells in the protein synthesis. Following to the evaluation of this Assessment Report, hydrolysed proteins were included in Annex I of Directive 91/414/EEC, as laid down in Commission Directive 2008/127/EC of 18 December 2008 and Commission Directive 2009/153/EC of 30 November 2009.

Based on results of read across studies, the lower 48 h EC50 value of 1.53 mg a.i./L (measured) for toxicity to Daphnia, has been considered further for hazard assessment of test substance, 'Steardimonium hydroxypropyl hydrolysed wool' as a conservative approach.