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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:
March 27, 2018 to March 29, 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:
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:
Under the study conditions, the 48 h EC50 of the test 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
Executive summary:

A study was conducted to determine the acute toxicity of test 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 test substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h, under static conditions. The concentrations of test substance in the test vessels were measured using the HPLC method. Based on the analysis, the mean measured concentrations of test 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 test 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).

Description of key information

Based on the study results, the 48 h EC50 of the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool', for toxicity in Daphnia magna was determined to be 2.44 mg/L (measured), i.e., equivalent to 1.53 mg a.i./L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.53 mg/L

Additional information

A study was conducted to determine the acute toxicity of test 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 test substance concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 mg/L for 48 h, under static conditions. The concentrations of test substance in the test vessels were measured using the HPLC method. Based on the analysis, the mean measured concentrations of test 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 test 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).