Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

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:

between 17 November 2008 and 3 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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

Principles of method if other than guideline:

Not Applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: LCE08082
Description: extremely pale yellow liquid
Date received: 5 September 2008
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids. Pre-study solubility work conducted indicated that the test material was readily water soluble and as such it was considered that the most suitable method of preparation for this material was as a direct solution in culture medium. Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode with m/z 511.4 (see Appendix 4 attached). However, as the test material is a mixture of various amino acid reaction products it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.


Analysis of the test preparations was carried out by TOC analysis. Water samples were taken from the control and each test group (replicates R1 - R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media). See section on analytical methods for TOC analysis details.

Duplicate samples were taken and stored at approximately -20 deg C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test the test material was dissolved directly in water.

An amount of test material (200 mg) was dissolved in reconstituted water with the aid of ultrasonication and the volume adjusted to 2 litres to give a 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18, 10, 5.6, 3.2, 1.8 and 1.0 mg/l.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) (see Appendix 2 - in attached section).

A positive control (Harlan Laboratories Ltd Project No: 0039/1094) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure conditions for the positive control were similar to those used in the definitive test.

The temperature was maintained at 21°C to 22°C. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol.

This was considered not to have affected the outcome or validity of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within test guideline specification.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 deg 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 suspension of algae (Chlorella sp.). 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 Water

The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Reconstituted Water

i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l


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-1. 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 CaCO3.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21 deg C to 22 deg C throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See attached Physico-Measurements for results.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See attached Physico-Measurements for results.

Salinity:

freshwater used.

Nominal and measured concentrations:

Following a preliminary range finding test, daphnids were exposed to teh test material over a range of nominal loading rates of 1.0,1.8,3.2,5.6,10,18,32,56 and 100 mg/L
The total Organic Carbon (TOC) analysis of the test preparations at 0 and 24 hours (fresh media) showed measured test concentrations in the range of less than the limit of quantification (LOQ) to 95 mg/L were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal test concentration only.

Based on the results of the range-finding test and in order to ensure that both EC50 and NOEC values were obtained the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.

Details on test conditions:

An amount of test material (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for 5 minutes and the volume adjusted to 500 ml to give a 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test material.
At the request of the Sponsor the test was conducted using semi-static test conditions. Therefore, the test media were freshly prepared at 0 and 24 hours prior to dosing.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
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.




As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20C to 22C with a photoperiod of 16 hours light 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.
At 48 hours (old media) the temperature in several of the test vessels was observed to be in excess of the 20 ± 1°C stated in the Protocol. This was considered to have no adverse effect on the outcome of the test as the temperatures were within the Test Guideline specification.
The control group was maintained under identical conditions but not exposed to the test material.
At the request of the Sponsor semi-static test conditions were employed in the test. 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: Not Applicable

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL 4.1 - 16 mg/l

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: No Immobilisation at this concentration

Remarks on result:

other: Not applicable

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: No immobilisation at this concentration

Remarks on result:

other: Not applicable

Details on results:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2 (in section any other information on results). The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (see attached).
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
24 >100 -
48 8.3 4.1 - 16

The No Observed Effect Concentrations after 24 and 48 hours exposure were 3.2 and 1.8 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 0.69 (SE = 0.34) and 1.7 (SE = 0.36) respectively.

Results with reference substance (positive control):

Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1094) during the positive control are given in Table 3 (in section any other information on results).

The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 2 and 3 (see in attached section)

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h)
EC50 (mg/l) 95% Confidence limits (mg/l)
3 > 3.2 -
24 1.0 0.90 - 1.2
48 0.78 0.68 - 0.88

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/l respectively.

The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 7.8 (SE = 1.7) and 12 (SE = 2.4) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.20).

Reported statistics and error estimates:

An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) and theEC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (Toxcalc 1999).
Probit analysis is used when two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	6	10
100	6	9

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0
3.2	0	0	0	0	2	1	3	15
5.6	4	3	7	35	5	9	14	70
10	4	3	7	35	7	7	14	70
18	5	3	8	40	8	7	13	65
32	1	1	2	10	8	9	17	85
56	4	5	9	45	7	10	17	85
100	4	1	5	25	9	9	18	90

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	0	1	5
1.0	0	0	0	0	7	3	10	50	10	8	18	90
1.8	0	0	0	0	10	9	19	95	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 8.3 mg/l with 95% confidence limits of 4.1 -16 mg/l. The No Observed Effect Concentration at 48 hours was 1.8 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in 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.

Methods. 

Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids. Pre-study solubility work conducted indicated that the test material was readily water soluble and as such it was considered that the most suitable method of preparation for this material was as a direct solution in culture medium. Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode with m/z 511.4. However, as the test material is a mixture of various amino acid reaction products it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to the test material over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours under semi-static test conditions at a temperature of 21°C to 22ºC. The number of immobilised Daphnia were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C to 22°C understatic test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was 8.3 mg/l with 95% confidence limits of 4.1 - 16 mg/l. The No Observed Effect Concentration was 1.8 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 24 hours (fresh media) showed measured test concentrations in the range of less than the limit of quantitation (LOQ) to 95 mg/l were obtained. Analysis of the test preparations at 24 and 48 hours (old media) showed measured test concentrations in the range of less than the LOQ to 92 mg/l were obtained.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.68 – 0.88 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Description of key information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 8.3 mg/l with 95% confidence limits of 4.1 -16 mg/l.  

The No Observed Effect Concentration at 48 hours was 1.8 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.3 mg/L