A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2002 - Februari 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 21st September 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification 2-Ethylhexylglucoside
Description Dark brown liquid
Batch 008143
Purity Solids: 63.5%; Water: 36.5%
Test substance storage At room temperature in the dark
Stability under storage conditions Not Indicated
Expiry date 16 February 2003
Density (kg/m3) 1150

Analytical monitoring:

yes

Details on sampling:

During the study samples were taken from all concentrations and the blank-control.

Sampling frequency
At the beginning and the end of an interval of 72 hours (nominal days 0 and 3), and at the start and the end of two 48 hours intervals (nominal days 8,10, 15 and 17).

Sample volume 10 ml.

Storage
Not applicable: the samples were transferred to the chemical department for pre-treatment immediately after sampling.

In addition, reserve samples were taken from all concentrations and the blank-control. These samples were stored in a deep-freeze for a maximum of three months after delivery of the draft
report, pending on the decision of the sponsor for additional analysis. Quantification of the exposure concentrations was based on measurement of 2-ETHYLHEXYLGLUCOSIDE concentrations, which were compared with calibration standards expressed as whole product. The methods of analysis are described in the appended Analytical Report.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS

The standard test procedures require generation of test solutions, which contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
Concentrations that disturb the test system will be excluded from testing (e.g. film of the test substance on the water surface or deposits of test substance on the bottom of the vessels).

The batch of 2-ETHYLHEXYLGLUCOSIDE tested was a dark brown liquid with a solid content of 63.5% and a water content of 36.5%. 2-ETHYLHEXYLGLUCOSIDE was soluble in water at the concentrations tested and no precipitation was observed. Preparation of the test solutions started with a stock solution prepared at 100 mg/I (63.5 mg a.i./L) in M7 medium. This solution was stirred for 10-25 minutes and subsequently diluted in M7 to reach the target concentrations. All test solutions were clear and colourless.

The test substance was biodegradable but analytical support of acute toxicity tests with Daphnia magna and fish showed that test concentrations were stable for at least 96 hours (Report KND 7S(a)/930392 and Report KND 7S(b)/930393, Huntington Research Centre Ltd., 26 August 1993).
The regime of renewal of test solutions was therefore set at complete renewal of the test solutions three times a week.

TEST CONCENTRATIONS
Range of the whole product 10, 1S, 32, 56 and 100 mg/I.
Range active ingredient Based on the actual contents of solids (63.5 %): 6.4, 11, 20,36 and 64 mg a.i./L
Controls Test medium without test substance or other additives (blank-control).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM

Species aphnia magna (Crustacea, Cladocera) (Straus, 1820).
Reason for selection This system has been selected as an internationally accepted species.
Validity of batch Frequent inspection of the cultures with respect to number of young, appearance of young and parental Daphnia and general feeding behaviour.
Characteristics For the test selection of young daphnids with an age of < 24 hours

BREEDING

Start of each batch With newborn animals, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium.
Maximum age of the cultures 4 weeks
Renewal of the medium Three times a week.
Temperature of medium 18-22°C, constant within ± 1 °C.
Feeding Daily, a suspension of fresh water algae (Chlorella pyrenoidosa ).
Medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.- P., 1990: Selenium deficiency in Crustacea. An ultra- structural approach to antennal damage in Daphnia magna, Straus).

Composition of medium M7:
The following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water (tap water purified by reverse
osmosis (Milli-RO); Millipore Corp., Bedford, Mass., USA) (mg/L):

Major chemicals:
CaC12·2H20 293.8
MgS04·7H2O 123.3
NaHC03 64.8
KCI 5.8
Trace elements (mgtl): B 0.125
Fe 0.05
Mn 0.025
Li, Rb and Sr 0.0125
Mo 0.0063
Br 0.0025
Cu 0.0016
Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003
Na2EDT A.2H2O 2.5
Macro nutrients (mg/L): Na2Si03·9H20 10
NaN03 0.27
KH2P04 0.14
K2HP04 0.18

Vitamins (µg/L): Thiamine 75.0
B12 1.0
Biotin 0.75

The hardness is 250 mg/L expressed as CaC03 and the pH will be 8.0 ± 0.2 after aeration.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

According to guideline

Hardness:

250 mg/L expressed as CaC03

Test temperature:

19.0 and 20.4°C

pH:

7.6 to 8.6

Dissolved oxygen:

> 6.0 mg/L

Salinity:

M7 Standard test medium

Conductivity:

M7 Standard test medium

Nominal and measured concentrations:

Nominal test item concentration: 0, 10, 18, 32, 56, 100 mg/L
Measured test item concentration: 0, 7.27, 15.1, 28.6, 51.9, 94.8 mg/L
Measured test item concentration based on active ingredient: 0, 4.62, 9.59, 18.2, 33.0, 60.2 mg/L

Details on test conditions:

TEST PROCEDURE AND CONDITIONS

Test type Semi-static
Renewal of the test media Three times a week (Monday, Wednesday and Friday).
Test duration 21 days
Test vessels Volume: 100 ml (8 x diameter of 4 cm), all-glass covered with a Perspex plate.
Test medium M7
Experimental design:
The daphnids were exposed individually from the start of the 21-day exposure period. Ten neonate <24 h old) daphnids were used per test concentration and twenty in the control group. Each vessel contained 5O mL of test solution.
Light 16 hours photoperiod daily.
Feeding
Daily, 0.25 ml of a Chlorella pyrenoidosa suspension containing 2x10^8 cells was added as feed for the daphnids. According to the intern NOTOX project 345105 the daily ration corresponds with 0.19 or 0.23 mg C/Daphnia/day. The 95% confidence interval is within the range of 0.1 to 0.2 mg C, which are the recommended values for daily feeding per daphnid in the reproduction test according to OECD Guideline 211.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

60.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

18.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

RESULTS

Measured concentrations of 2-ETHYLHEXYLGLUCOSIDE in water samples

The results of the analysis of the samples taken during the test period are listed in Table 13 and Table 14 in the appended Analytical Report. The concentrations of 2-ETHYLHEXYLGLUCOSIDE were based on three separate peaks in the chromatograms (defined as peak 1,2 and 3). The area of each of the three test substance peaks (divided by the internal standard peak area) measured in the samples was compared to analytical standards expressed in concentrations of the whole product (not corrected for 63.5% solids). Quantification was performed by Gas Chromatographic method with Mass Spectrometry detection (GC-MS) using an extracted ion chromatogram from the total ion current.

The table below (Table 1) shows the concentrations measured in the samples taken during the test period. When based on peak 1 and 2, there was consistency between 2-ETHYLHEXYL-GLUCOSIDE concentrations measured in the freshly prepared solutions at the various renewals. Hence, the analytical support confirmed the repeatability of the preparation of the test concentrations.
Mainly based on peak 1 the concentrations measured remained stable during the periods between renewals except for day 17, when the two lowest concentrations decreased by 70 and 90 %, respectively. The higher concentrations did not decrease by more than 20%. This relatively high decrease at the lower concentrations was probably due to the presence of young daphnids and algae, which were more abundant than in preceding renewal periods.

Peaks 2 and 3 were less stable and more difficult to analyse. Measured concentrations based on peak 2 were below 80% of nominal on days 3,10 and 17, whereas measured concentrations based on peak 3 were below 80% of nominal on all days except day 0 (see table 2 of the analytical report).

Based on these results, toxicity parameters were calculated using the average exposure concentrations based on peak 1. Calculated average exposure concentrations are shown in Table 1. Based on this evaluation the actual range of test concentrations was 7.27, 15.1, 28.6, 51.9 and 94.8 mg/L. Expressed for 63.5% solids, the actual range was 4.62, 9.59, 18.2, 33.0 and 60.2 mg a.i./L.


Parental mortality

Cumulative mortality is presented in Table 2. Two out of twenty parental daphnids died during the test period in the blank control. At average exposure concentrations of 7.27 mg/L (4.62 mg a.i./L), 15.1 mg/L (9.6 mg a.i./L) and 94.8 mg/L (60.2 mg a.i./L) , one parental daphnid died during the test period, while no daphnids died at 28.6 mg/L (18.2 mg a.i./L) and 51.9 mg/L (33.0 mg a.i./L).
Therefore, mortality was not treatment related.

Reported statistics and error estimates:

Statistical analysis

The results of reproduction were tested for normality and for homogeneity of variance. The data were statistically tested using the Tukey test, Dunnett test and Bonferroni t-test. In addition, the data were tested using a general linear model under SAS.
The overall threshold level of effect and the overall NOEC and LOEC were determined on basis of these statistics. The EC10 and NEC (reproduction) were calculated.

Parameter	Concentration 2-ETHYLHEXYLGLUCOSIDE (mg/L)
	Whole test product (mg/L)		Active ingredient (mg a.i./L)
	Nominal	Actual
21-day NOEC for survival	100	94.8	60.2
21-day EC50 for reproduction	121 ± 12.7	115 ± 12.4	73.0 ± 7.87
21-day LOEC for reproduction	56	51.9	33.0
21-day NOEC for reproduction	32	28.6	18.2
21-day LOEC for body length	100	94.8	60.2
21-day NOEC for body length	56	51.9	33.0
Overall LOEC	56	51.9	33.0
NEC (95%-confidence interval)	42.2 (21.9 – 48.9)	38.5 (18.8 – 45.0)	24.5 (11.9 – 28.6)
Overall NOEC	32	28.6	18.2

Validity criteria fulfilled:

yes

Conclusions:

ACCEPTABILITY OF THE TEST
1. Mortality of parental daphnids in the controls did not exceed 20%.
2. The average cumulative number of young per female in the controls after 21 days was >60 (192.3 ± 16.5).
3. Incidentally, the deviation in pH values is in confirmation with the OECD guideline 211, i.e.not greater than 1.5 units.
4. The chemical analysis showed that the concentrations of 2-ETHYLHEXYL-GLUCOSIDE were best interpretable when based on peak 1. These concentrations remained above 80% of initial values during the measured renewal periods and the mean values ranged between 89 and 95 % of the nominal values in the three highest treatments, which were also biologically active. Hence, valid values for effect parameters could be calculated when based on geometric mean concentrations of this peak.

Executive summary:

Daphnia magna, reproduction test with 2-ETHYLHEXYLGLUCOSIDE.

This study investigated the effects of 2-ETHYLHEXYLGLUCOSIDE on survival and reproductive performance in Daphnia magna during 21 days of exposure. The study design was based on the OECD guideline No. 211 :"Daphnia magna reproduction test", adopted 21 5t September 1998.

The batch of 2-ETHYLHEXYLGLUCOSIDE tested was a dark brown liquid with a solid content of 63.5% and a water content of 36.5%. 2-ETHYLHEXYLGLUCOSIDE was soluble in water at the

concentrations tested and no precipitation was observed. A stock solution was prepared in M7 medium at 100 mg/l. This solution was diluted in M7 medium to reach the target concentrations.

All test solutions were clear and colourless.

The experimental set-up included 10 vessels per test concentration and 20 vessels for the control group (blank-control), each containing one neonate «24h old) Daphnia magna in 50 ml test medium. The nominal concentrations of 2-ETHYLHEXYLGLUCOSIDE tested were 10, 18, 32, 56 and 100 mg/l. Based on the actual contents of solids (63.5 %) the nominal concentrations of the active ingredient were: 6.4, 11, 20, 36, 64 mg a.i.!1. The study duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with Chlorella pyrenoidosa (approximately 0.6*10^6 cells/ml/day). At the start of the test and every workday the condition of the parental daphnids was recorded and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured. During the study samples for analysis were taken from freshly prepared solutions on nominal days 0, 8, and 15, and from 48 or 72-hour old solutions on nominal days 3, 10 and 17.

2-ETHYLHEXYLGLUCOSIDE consisted of three peaks. Chemical analysis showed that peak 1 was the most stable throughout the test period. Therefore, the toxicity parameters were calculated with the geometric means of measured 2-ETHYLHEXYLGLUCOSIDE concentrations based on peak 1. In addition toxicity parameters were also recalculated to the active ingredient based on the solid content (63.5 %).

Mortality of the parental daphnids in the controls did not exceed 20% at the end of the test. The mean number of living offspring per control parent at the end of the test was >= 60 (i.e. 192.3 ± 16.5). Temperature and dissolved oxygen concentrations remained within the ranges prescribed by the protocol and the test guidelines. Deviations in pH remained within the range prescribed by the OECD guideline. At average exposure concentrations of 7.27 mg/l (4.62 mg a.i./L), 15.1 mg/l (9.6 mg a.i./L) and 94.8 mg/l (60.2 mg a.i./L), one parental daphnid died during the test period, while no daphnids died at 28.6 mg/l (18.2 mg a.i./L) and 51.9 mg/L (33.0 mg a.i./L). Therefore, mortality was not treatment related.

Statistical analysis of the reproduction data revealed that the reproductive capacity of daphnids exposed to 2-ETHYLHEXYLGLUCOSIDE was not affected up to an average exposure concentration of 28.6 mg/L (18.2 mg a.i./L). A significant decrease in reproductive capacity was found at 51.9 and 94.8 mg/L, corresponding to 33.0 and 60.2 mg a.i./L (Dunett test and Tukey test: p<0.05).

There was no statistical difference between body lengths of the parental daphnids at average exposure concentrations up to and including 51.9 mg/L or 33.0 mg a.i./L and the controls according to the Tukey test and Bonferroni t-test (p>0.05). At 94.8 mg/L (60.2 mg a.i./L), the length of the daphnids was significantly reduced (Tukey test and Bonferroni test, p<0.05).

Parameter	Concentration 2-ETHYLHEXYLGLUCOSIDE (mg/L)
	Whole test product (mg/L)		Active ingredient (mg a.i./L)
	Nominal	Actual
21-day NOEC for survival	100	94.8	60.2
21-day EC50 for reproduction	121 ± 12.7	115 ± 12.4	73.0 ± 7.87
21-day LOEC for reproduction	56	51.9	33.0
21-day NOEC for reproduction	32	28.6	18.2
21-day LOEC for body length	100	94.8	60.2
21-day NOEC for body length	56	51.9	33.0
Overall LOEC	56	51.9	33.0
NEC (95%-confidence interval)	42.2 (21.9 – 48.9)	38.5 (18.8 – 45.0)	24.5 (11.9 – 28.6)
Overall NOEC	32	28.6	18.2

Description of key information

A long-term daphnia test has been performed with ethylhexylglucoside according to OECD TG 211 under GLP conditions.

The test solutions were refreshed three times a week and the exposure concentrations used for the dose response are based on geometric mean concentrations as observed for peak 1 (three peaks were observed).

A NOEC for reproduction based on geometric mean measured concentrations was observed of 18.2 mg active ingredient/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

Based on Active ingredient

Effect concentration:

18.2 mg/L

Additional information

A long term daphnia test with 2 -ethylhexylglucoside was performed according to OECD guideline No. 211 :"Daphnia magna reproduction test", adopted 21 5t September 1998.

The test substance was soluble in water and test solutions were prepared via dilution of a stock solution in M7 test medium. All test solutions were clear and colourless.

The nominal test concentrations were 10, 18, 32, 56 and 100 mg/L. Based on the test item and 6.4, 11, 20, 36, 64 mg/L based on active ingredient (63.5%). The study duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with Chlorella pyrenoidosa (approximately 0.6*10^6 cells/ml/day). At the start of the test and every workday the condition of the parental daphnids was recorded and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured. During the study samples for analysis were taken from freshly prepared solutions on nominal days 0, 8, and 15, and from 48 or 72-hour old solutions on nominal days 3, 10 and 17. Geometric mean concentrations were used for the calculation of the dose response.

Mortality of the parental daphnids in the controls did not exceed 20% at the end of the test. The mean number of living offspring per control parent at the end of the test was >= 60 (i.e. 192.3 ± 16.5). Temperature and dissolved oxygen concentrations remained within the ranges prescribed by the protocol and the test guidelines. Deviations in pH remained within the range prescribed by the OECD guideline. At average exposure concentrations of 7.27 mg/l (4.62 mg a.i./L), 15.1 mg/l (9.6 mg a.i./L) and 94.8 mg/l (60.2 mg a.i./L), one parental daphnid died during the test period, while no daphnids died at 28.6 mg/l (18.2 mg a.i./L) and 51.9 mg/L (33.0 mg a.i./L). Therefore, mortality was not treatment related.

Statistical analysis of the reproduction data revealed that the reproductive capacity of daphnids exposed to 2-ETHYLHEXYLGLUCOSIDE was not affected up to an average exposure concentration of 28.6 mg/L (18.2 mg a.i./L). A significant decrease in reproductive capacity was found at 51.9 and 94.8 mg/L, corresponding to 33.0 and 60.2 mg a.i./L (Dunett test and Tukey test: p<0.05).

There was no statistical difference between body lengths of the parental daphnids at average exposure concentrations up to and including 51.9 mg/L or 33.0 mg a.i./L and the controls according to the Tukey test and Bonferroni t-test (p>0.05). At 94.8 mg/L (60.2 mg a.i./L), the length of the daphnids was significantly reduced (Tukey test and Bonferroni test, p<0.05).

Parameter	Concentration 2 -ETHYLHEXYLGLUCOSIDE (mg/L)
	Whole test product (mg/L)		Active ingredient (mg a.i./L)
	Nominal	Actual
21-day NOEC for survival	100	94.8	60.2
21-day EC50 for reproduction	121 ± 12.7	115 ± 12.4	73.0 ± 7.87
21-day LOEC for reproduction	56	51.9	33.0
21-day NOEC for reproduction	32	28.6	18.2
21-day LOEC for body length	100	94.8	60.2
21-day NOEC for body length	56	51.9	33.0
Overall LOEC	56	51.9	33.0
NEC (95%-confidence interval)	42.2 (21.9 – 48.9)	38.5 (18.8 – 45.0)	24.5 (11.9 – 28.6)
Overall NOEC	32	28.6	18.2