Potassium Lauroyl Wheat Amino Acids

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 22, 2018 to May 24, 2018

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:

yes

Remarks:

The age of Daphnia was 24 h 13 minutes; however as per the opinion of the study director this would not have affected the validity or integrity of this study, as there was no mortality even in the control group and the all validity criteria were met.

GLP compliance:

yes (incl. QA statement)

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 48 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 48 h. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

Vehicle:

yes

Remarks:

Elendt M4 medium

Details on test solutions:

Preliminary solubility tests indicated that the sample was soluble after ultrasonication at >100mg/L in water. 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: 21 May 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:

20.4 – 21.2°C

pH:

7.03 - 7.52

Dissolved oxygen:

8.71 – 9.53 mg/L

Nominal and measured concentrations:

Nominal: 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L
Mean measured: 0 (control), 1.796, 3.436, 10.400, 18.337 and 46.888 mg/L

Details on test conditions:

Test methods and 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 h 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 >100mg/L.

(B) Definitive test method: Test period: 22 to 24 May 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: 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L
Mean measured concentrations: 0 (control), 1.796, 3.436, 10.400, 18.337 and 46.888 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 Hours: 7.45 (Required: 6-9)
pH range in control and test concentrations throughout test: 7.03 – 7.52
Initial Dissolved Oxygen at 0 Hours: 9.49mg/L
DO range in control and test concentrations throughout test: 8.71 – 9.53mg/L (Required: ≥3mg/L)
Temperature range within incubator throughout test: 20.4 - 21.2 °C (Required: 20±2°C maintained to ±1°C)
Illumination: 16 Hours Light, 8 Hours 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 48 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 hours and pooled replicate flasks for 48 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 hour 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.

Study plan deviation
- Test species: states that the Daphnia “will be <24-hour old juveniles”. The Daphnia in the test were isolated at 14:17 21 May 2018 and the test was initiated at 14:30 22 May 2018 meaning the Daphnia were 24 hours and 13 minutes old.
- There was no mortality or no behavioural abnormalities within the control units. A 13 minute deviation in Daphnia age is not significant. Therefore we can attribute any mortality in the test concentrations to be due to the test substance. All necessary validity criteria was met and in the opinion of the study director, this would not have affected the validity or integrity of this study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: or 69.8 mg a.i./L; confidence limits not possible to determined

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: or 34.9 mg a.i./L; determined by Steel’s Many-One Rank Test

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.

Reported statistics and error estimates:

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.

Results:

Analytical results

Concentration of test substance (mg/L) determined by HPLC UV analysis

 

Nominal Concentration (mg/L)	Concentrations of test substance (mg/L) calculated from measured active ingredient concentrations at the following sampling times
	0hrs	48hrs	Mean measured
6.25	5.669	1.515	1.796
12.5	12.527	1.216	3.436
25	25.289	16.309	10.400
50	50.537	22.809	18.337
100	107.129	80.424	46.888

 

 

Average recoveries of test substance (mg/L), percentage recovery compared to nominal concentrations.

 

Nominal Concentration (mg/L)	Concentrations of test substance (mg/L) calculated from measured active ingredient concentrations at the following sampling times
	0hrs	48hrs	Mean measured
6.25	90.711	24.235	28.736
12.5	100.215	9.727	27.485
25	101.158	65.237	41.599
50	101.074	45.618	36.673
100	107.129	80.424	46.888

The analytical measurements of the freshly prepared exposure solutions indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement of the aged exposure solutions showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The mean measured concentrations of test substance were determined to be 0 (control), 1.796, 3.436, 10.400, 18.337 and 46.888 mg/L respectively. Due to the poor recovery, all effect concentrations were reported as nominal concentrations of the test substance.

Test 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
6.25	1.796	0	0	0	0
12.5	3.436	1	2	5	10
25	10.400	0	1	0	5
50	18.337	0	0	0	0
100	46.888	9	10	45	50

Observations

Nominal Concentration mg/L	Mean measured concentration mg/L	Recorded observations
		24 h	48 h
0 (Control)	0 control	20NS	20NS
6.25	1.796	20NS	19NS1TM
12.5	3.436	19NS	18NS
25	10.400	20NS	19NS
50	18.337	20NS	20NS
100	46.888	11NS	8NS2MS

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	>100	*
48	>100	*
Statistical methods used in ToxCalc v5.0: Linear Interpolation. *Confidence limits not possible to determine. ^$Normality of distribution and equality of variances could not be confirmed. All concentrations of the test substance are reported as nominal as received.

Discussion

The definitive test conducted from 22 to 24 May 2018 was performed according to the OECD 202 2004 guideline and met all validity criteria. The 24-h EC₅₀ and 48 -h EC₅₀ of test substance to Daphnia magna were >100 mg/L and >100 mg/L respectively (both determined by Linear Interpolation). Graphical representations of the 24 -h and 48 -h EC₅₀ values are given in Graphs 1 and 2 respectively (For graphs, kindly refer to attached background material section of IUCLID). The 0 to 24-h NOEC and LOEC were 50 mg/L and 100 mg/L respectively (determined by Steel’s Many-One Rank Test). The 0 to 48 -h NOEC and LOEC were 50 mg/L and 100 mg/L respectively (determined by Steel’s Many-One Rank Test). A graphical representation of the 48 -h dose response plot is given in Graph 3. The Daphnia were examined at 24 -h and 48 -h for abnormal behaviour during the determination of immobility. Abnormalities were observed during the test. No concentration immobilised all twenty Daphnia after 48 -h. The highest concentration where no immobilisation occurred was 50 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 full water quality measurements 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. Analytical measurements of the freshly prepared exposure solutions indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement of the aged exposure solutions showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. All effect concentrations are reported as nominal concentrations of the test substance.The definitive tests conducted on 04 to 06 April 2018 was not reported as a response was seen at 100 mg/L and 9 to 11 May 2018 was not reported as a NOEC could not be determined.

 

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 >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal).

Executive summary:

A study was conducted to determine the acute toxicity of test substance, 'potassium lauroyl wheat amino acids' (active: 69.8%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. A preliminary (range-finding) test was conducted at concentrations of 0 (control), 0.1, 1, 10 and 10 0mg/L. Data from the preliminary test identified the 48-hour EC50 to be >100mg/L. Therefore in the main study, four replicates of twenty test organisms were exposed to each nominal test substance concentrations of 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L for 48 h, under static conditions. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 48 h exposure period using HPLC.Analytical measurements at 0 h indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement at end of 48 h showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The meanmeasured concentration of the test substances were determined to be 0 (control), 1.796, 3.436, 10.400, 18.337 and 46.888 mg/L respectively. Due to poor recovery, all effect concentrations were therefore reported as nominal concentrations of the test substance.Abnormal behaviour and the number of immobilised Daphnia were both recorded after 24 and 48-h exposure periods. None of the test concentration showed signs of abnormal behaviour or immobilisation in all twenty Daphnia after 48-h. The highest concentration where no immobilisation or abnormal behaviour occurred was 50 mg/L (nominal). None (0 %) of the twenty control Daphnia maintained in dilution water were immobilised and none were trapped in the meniscus during the study. As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L and the percentage of immobilisation did not exceed 10%, all validity criteria were considerd to be fulfilled according to the guideline. Based on immobility compared to the control (p <0.05), the 48 h NOEC values was determined to be 50 mg/L (i.e., equivalent to 34.9 mg a.i./L; nominal) and the 48 h EC50 was calculated to be >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal). Under the study conditions, the 48 h EC50 of the test substance for toxicity in Daphnia magna was determined to be >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal) (Chemex, 2018).

Description of key information

Based on study results, the 48 h EC50 of the test substance for toxicity in Daphnia magna was determined to be >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

69.8 mg/L

Additional information

A study was conducted to determine the acute toxicity of test substance, 'potassium lauroyl wheat amino acids' (active: 69.8%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. A preliminary (range-finding) test was conducted at concentrations of 0 (control), 0.1, 1, 10 and 10 0mg/L. Data from the preliminary test identified the 48-hour EC50 to be >100mg/L. Therefore in the main study, four replicates of twenty test organisms were exposed to each nominal test substance concentrations of 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L for 48 h, under static conditions. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 48 h exposure period using HPLC.Analytical measurements at 0 h indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement at end of 48 h showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The meanmeasured concentration of the test substances were determined to be 0 (control), 1.796, 3.436, 10.400, 18.337 and 46.888 mg/L respectively. Due to poor recovery, all effect concentrations were therefore reported as nominal concentrations of the test substance.Abnormal behaviour and the number of immobilised Daphnia were both recorded after 24 and 48-h exposure periods. None of the test concentration showed signs of abnormal behaviour or immobilisation in all twenty Daphnia after 48-h. The highest concentration where no immobilisation or abnormal behaviour occurred was 50 mg/L (nominal). None (0 %) of the twenty control Daphnia maintained in dilution water were immobilised and none were trapped in the meniscus during the study. As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L and the percentage of immobilisation did not exceed 10%, all validity criteria were considerd to be fulfilled according to the guideline. Based on immobility compared to the control (p <0.05), the 48 h NOEC values was determined to be 50 mg/L (i.e., equivalent to 34.9 mg a.i./L; nominal) and the 48 h EC50 was calculated to be >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal). Under the study conditions, the 48 h EC50 of the test substance for toxicity in Daphnia magna was determined to be >100 mg/L (i.e., equivalent to 69.8 mg a.i./L; nominal) (Chemex, 2018).