Sodium 5-oxo-1-palmitoyl-L-prolinate

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:

The study was conducted between 4 November 2008 and 21 September 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:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: LCA08010
Description: greenish brown turbid viscous liquid
Batch number: 0729900003
Date received: 2 July 2008
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis.

Water samples were taken from the control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) (see Appendix 3 - in attached section).

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

Vehicle:

no

Details on test solutions:

Amounts of test material (20, 36, 64, 112 and 200 mg) were each separately added to the surface of 20 litres of reconstituted water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/l loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/l loading rate WAFs.

Controls:
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 temperature 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
daphnids throughout the duration of the test and that the temperature were within test guideline specification.

Evidence of undissolved material :
Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions or particles of test material
present.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species
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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 Appendix 4 in section any other information on methods

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 Appendix 4 in section any other information on methods

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l.

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

Details on test conditions:

Amounts of test material (20, 200 and 2000 mg) were each separately added to the surface of 20 litres of reconstituted water to give the 1.0, 10 and 100 mg/l loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs.

Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions or particles of test material present.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21ºC to 22ºC 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.
S
everal temperatures at 0 and 48 hours were 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.

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 22ºC 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.

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.

Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

No. of organisms per vessel:
10 daphnids were placed in each test and control vessel at random, in the test preparations

The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Remarks:

Effective Loading Rate

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 2.9 - 3.7 mg/l.

Duration:

48 h

Dose descriptor:

EL50

Remarks:

Effective Loading Rate

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 2.0 - 2.4 mg/l

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: Not stated

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 (see section
any other information on results). The relationship between percentage immobilisation and loading rate at 24 and 48 hours is given in Figures 1 and 2 (see attached)

Analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 and 48 hours gave the following results:

Time (h) EL*50 (mg/l Loading Rate WAF) 95% Confidence limits
(mg/l Loading Rate WAF)
24 3.3 2.9 – 3.7
48 2.2 2.0 – 2.4

The No Observed Effect Loading rates after 24 and 48 hours exposure were 1.8 and 1.0 mg/l loading rate WAF respectively. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of response curve at 24 and 48 hours.

Results with reference substance (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 4 (see section any other information on results). The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 3 and 4 (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 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 values and associated confidence limits at 24 and 48 hours and the slope of the response curves and standard errors 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.

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table1.

Whilst no immobilisation was observed at 1.0 mg/l loading rate WAF, significant immobilisation was observed at both 10 and 100 mg/l loading rate WAF.

Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	10	10
100	10	10

Table 2              Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (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	2	1	3	15
3.2	4	5	9	45	10	10	20	100
5.6	10	10	20	100	10	10	20	100
10	10	10	20	100	10	10	20	100

Table 3              Vortex Depth Measurements at the Start and End of the First Mixing Period

	Nominal Loading Rate (mg/l)
	Control		1.0		1.8
	*	+	*	+	*	+
Height of Water Column (cm)	34.0	34.0	33.5	33.5	34.5	34.5
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	3.2		5.6		10
	*	+	*	+	*	+
Height of Water Column (cm)	33.0	33.0	33.0	33.0	33.0	33.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

 

 

	Nominal Loading Rate (mg/l)
	Control		1.0		1.8
	*	+	*	+	*	+
Height of Water Column (cm)	32.5	32.5	33.0	33.0	33.5	33.5
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	3.2		5.6		10
	*	+	*	+	*	+
Height of Water Column (cm)	32.5	32.5	33.0	33.0	33.0	33.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

 

Table 4              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 2

*= Start of mixing period

+= End of mixing period

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 EL*50 value of
2.2 mg/l loading rate WAF with 95% confidence limits of 2.0 – 2.4 mg/l loading rate WAF.

The No Observed Effect Loading rate at 48 hours was 1.0 mg/l loading rate WAF.

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 mixture of components. Given this and also the poor solubility of the test material in water it was considered that the most suitable method of preparation for this material was as a Water Accommodated Fraction (WAF).

Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode at 354 and 396 m/z. However, as the test material is a mixture of components 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 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/l for 48 hours at a temperature of 22°C under semi-static test conditions. 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 under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EL*₅₀ for the test material to Daphnia magna based on nominal loading rates was 2.2 mg/l loading rate WAF with 95% confidence limits of 2.0 – 2.4 mg/l loading rate WAF. The No Observed Effect Loading rate was 1.0 mg/l loading rate WAF.

Total Organic Carbon (TOC) analysis of the test preparations was performed on the fresh test media at 0 and 24 hours and on the old/expired test media at 24 and 48 hours. Overall the results showed a concentration dependant increase in TOC with increasing loading rate in the range of less than the limit of quantitation (LOQ) to 4.87 mg C/l.

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 loading rates 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.

*EL = Effective Loading Rate

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 EL*50 value of 2.2 mg/l loading rate WAF with 95% confidence limits of 2.0 – 2.4 mg/l loading rate WAF.  

The No Observed Effect Loading rate at 48 hours was 1.0 mg/l loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.2 mg/L