Bis(2-ethylhexyl) malate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th November 2017 to 30 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) malate

EC Number:

260-070-5

EC Name:

Bis(2-ethylhexyl) malate

Cas Number:

56235-92-8

Molecular formula:

C20H38O5

IUPAC Name:

1,4-bis(2-ethylhexyl) 2-hydroxybutanedioate

Test material form:

liquid

Details on test material:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)
Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5

Specific details on test material used for the study:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Test item storage: At room temperature
Purity/Composition correction factor: No correction factor required
Organic carbon (w%): 67.0%
Test item handling: No specific handling conditions required
Stability at higher temperatures: Stable but no data and so prefer not to heat
Chemical name (IUPAC), synonym or trade name: Diethylhexyl Malate
CAS number: 56235-92-8
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 5).

Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the limit concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Dermol DOM tested was a clear colourless to pale yellow liquid with a purity >95% which was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 3-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 55 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning. After siphoning, a floating layer of undissolved material was observed in the SS. Hence, the SS was stabilised for another 2 hours and subsequently, siphoned over glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.
The water solubility at 20°C was determined to be 0.22 mg/L, using the low-stirring method (Test Facility Study No. 20136808).

Test Concentrations

Dermol DOM
Solutions containing 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L.

Control
Test medium without test item or other additives.

Replicates
3 replicates of each test concentration,
6 replicates of the control and undiluted SS,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

°C

Nominal and measured concentrations:

Nominal: [insert concenterations] mg/l [Loading rate]
Measured mean: [insert concentrations] mg/l

Details on test conditions:

Test Procedure and Conditions
Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass, containing 50 mL of test solution
Medium M2
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity of 88 µE.m-2.s-1.
Incubation Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

[State positive control substance]

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Based on TWA concentrations

Effect conc.:

> 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Based on TWA concentrations and statistical significance

Effect conc.:

< 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Based on TWA concentrations

Effect conc.:

> 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Based on TWA concentrations and biological significance

Effect conc.:

ca. 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Based on TWA concentrations and statistical significance

Effect conc.:

< 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Based on TWA concentrations and biological significance

Effect conc.:

< 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Based on TWA concentrations

Effect conc.:

< 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Based on TWA concentrations

Effect conc.:

> 0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Measured Test Item Concentrations
The results of analysis of the samples taken during the test are described in Table 2 analytical report.
Samples taken from the undiluted SS prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was 2.2 mg/L, which decreased to 0.019% of initial at the end of the test. The concentrations measured in samples with algae were comparable with the concentrations measured in the samples without algae. Based on these results, effect parameters were based on the Time Weighted Average (TWA) concentration (see Table below).
The measured concentration at the start of the test exceeded the water solubility limit of 0.22 mg/L (Test Facility Study No. 20136808). At the end of the exposure period, the measured concentration decreased below the water solubility of 0.22 mg/L. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium with a TWA concentration of 0.097 mg/L. It should be noted that a small response was estimated in the control samples at 24 and 48 hours of exposure. However, since an even higher concentration was estimated in one of the quality control sample, and the contribution to the test concentration measured at 24 hours was negligible, it was decided not to correct the final exposure concentrations.

Measured Concentrations Versus Percentage of the Saturated Solution

Dermol DOM %SS prep. Measured concentration (mg/L) TWA conc. (mg/L)
at a loading rat of 100 mg/L
t=0h t=24h t=72 h
100 2.15 0.0373 0.00043 0.097

Mean Cell Densities
Figure 1 (attached) shows growth curves at the control and undiluted SS of Dermol DOM. The individual and group mean cell densities measured at 24h intervals are given in Table 7 (attached).

Inhibition of Growth Rate and Inhibition of Yield
Table 2 below shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 3 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 4 (see attached Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3 (attached).
Growth rate and yield at the limit concentration were statistically significantly inhibited by 2.6 and 13%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 0.097 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the undiluted SS when compared to the control.

Table 2
Growth Rate And Percentage Inhibition For The Total Test Period

Dermol DOM %SS prep.
at a loading rate of 100 mg/L Mean Std. Dev. n %Inhibition
Control 1.844 0.0095 6
1.0 1.844 0.0096 3 -0.013
10 1.820 0.0368 3 1.3
100 (0.097) 1.795 0.0193 6 2.6#
# – effect statistically significant but biologically not relevant (<10%), ( ) – TWA concentration (mg/L)

Table 3
Growth Rate And Percentage Inhibition At Different Time Intervals

Dermol DOM %SS prep.
at a loading rate of 100 mg/L n 0 – 24 h 24 – 48 h 48 – 72h
Mean %Inhibition Mean %Inhibition Mean %Inhibition
Control 6 2.240 1.767 1.524
100 (0.097) 6 2.042 8.8 1.696 4.0 1.647 -8.1
( ) – TWA concentration (mg/L)

Table 4
Yield And Percentage Inhibition For The Total Test Period

Dermol DOM %SS prep.
at a loading rate of 100 mg/L Mean Std. Dev. n %Inhibition
Control 251.5 7.19 6
1.0 251.6 7.21 3 -0.063
10 235.1 25.26 3 6.5
100 (0.097) 217.6 13.03 6 13*
* – effect statistically significant
( ) – TWA concentration (mg/L).
 
Determination of Effect Concentrations
Table 5 shows the effect parameters based on the TWA concentration.

Table 5
Effect Parameters

Parameter (mg/L) NOEC* NOEC** EC10 EC50
Growth rate <0.097 0.097 >0.097 >0.097
Yield <0.097 <0.097 <0.097 >0.097
* - based on statistical significance, ** - based on biological relevance

Experimental Conditions
Table 6 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Table 6
pH Levels Recorded During the Test

Dermol DOM %SS prep.
at a loading rate of 100 mg/L pH
t=0h t=72h
Control 8.1 8.2
100 (0.097) 8.1 8.2
( ) – TWA concentration (mg/L)

Results with reference substance (positive control):

Included in attached Appendix 4.

Reported statistics and error estimates:

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 253. The test fulfils this validity criterion.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the tested concentrations of Dermol DOM.
The EC50 for growth rate inhibition (72h-ERC50) and yield inhibition (72h-EYC50) exceeded the solubility limit of Dermol DOM in test medium, i.e. exceeded a TWA concentration of 0.097 mg/L.
The 72h-NOEC for growth rate inhibition was below the TWA concentration of 0.097 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was 0.097 mg/L (TWA concentration) based on biological relevance.
The 72h-NOEC for yield inhibition was below the TWA concentration of 0.097 mg/L based on both statistical significance and biological relevance.

Executive summary:

The objectiveofthe study was to evaluate Dermol DOM for its ability to generate toxic effects inRaphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata)during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Dermol DOM tested was a clear colourless to pale yellow liquid with a purity >95% which was not completely soluble in test medium at the loading rate initially prepared.A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to a SS prepared at a loading rate of 100 mg/L in the limit test. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the SS in a combined range-finding test. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the undiluted SS prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was 2.2 mg/L, which decreased to 0.019% of initial at the end of the test. The concentrations measured in samples with algae were comparable with the concentrations measured in the samples without algae. Based on these results, effect parameters were based on the Time Weighted Average (TWA) concentration of 0.097 mg/L.

Growth rate and yield at the limit concentration were statistically significantly inhibited by 2.6 and 13%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 0.097 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters based on TWA concentrations and obtained in this study are summarized in the table below.

Parameter (mg/L)	NOEC*	NOEC**	EC10	EC50
Growth rate	<0.097	0.097	>0.097	>0.097
Yield	<0.097	<0.097	<0.097	>0.097

* - based on statistical significance, ** - based on biological relevance