D,L-DIETHYL TARTRATE

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 July 2017 to 4 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 201 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 4, 5 and 6 July 2016 / Signed on 10 January 2017

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) at the start (t=0 h) and the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water was added. Then 1019 mg test item were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1 L of stock solution (approximate headspace: 3-4 cm) and thereafter was closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 20% vessel depth from the top to the bottom of the vessel. After 1 hour of gentle stirring at room temperature, the content of the vessel was allowed to stand undisturbed for at least 5 min before use. Then the stock solution was diluted with test water as necessary and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells.mL-1 per vessel. All the preparation was performed under strict sterile conditions. At the start of the test, the solution was observed to be clear and colourless.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes.
- Stock Culture: Algae stock cultures were started by inoculating growth medium (= test water) 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 23 ± 2 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

No data

Test temperature:

22.6-23.6 °C (average value: 23.3 °C)

pH:

7.66 to 9.47

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 60, 106, 189, 337 and 600 mg/L
Corresponding average exposure concentrations (100% test item): 24.39, 31.74, 76.24, 261.45 and 507.94 mg/L (see table 6.1.5/2 in "Any other information on results incl. tables")

Details on test conditions:

TEST SYSTEM
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, filled with 50 mL of test medium.
- Volume: Volume of 50 mL of algal suspension per replicate
- Initial cells density: An initial cell density of 5000 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additionally, a replicate of each test concentration with algae was prepared for chemical analyses.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Original medium of OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Light intensity of 4440-8880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
pH: At the start (t=0 h) and every day thereafter until the end of the test in one vessel per concentration and the control.
Temperature of medium: Measured continuously in the growth chamber, over the study period.
Light intensity: Light intensity was measured at t=0 h at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Range finding study: A range-finding test was conducted to determine the range of concentrations for the definitive test. In this study, algal cells were exposed to a series of nominal test concentrations of 0, 50, 500, 5000 and 50000 mg/L for 72 h. Algal cell densities at 72 h (expressed as density of algal cells/mL^-1x10^4).): 97.9, 94.9, 5.6, 0.8 and 0, respectively.
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations (spaced by a factor of approximately 1.78): 60, 106, 189, 337 and 600 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

369.317 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 339.023 – 403.822 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

124.814 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 91.822 – 152.853 mg/L

Details on results:

Water quality parameters and environmental conditions throughout the test:
The temperature in the incubator was situated between 22.6 and 23.6 °C throughout the test (average value: 23.3 °C). The mean light intensity was of 5212 lux (range: 4845-5719 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23 ± 2 °C, constant within 2 °C; light intensity: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area). Moreover, the results of micro-organisms counting revealed the absence of microbial contamination in test solutions.

Analytical results (see Table 6.1.5/2 in "Any other information on results incl. tables"):
Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine maintenance of actual concentrations of the test item. Chemical analyses revealed severe test item losses, especially at the lowest test concentrations (where toxicity was less pronounced or not observed). This could suggest metabolism effects of the test item by P. subcapitata at the lower concentrations, and hydrolysis at the higher concentrations (as confirmed by decreases in pH values). Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations for the present study.

Results with reference substance (positive control):

On February 14, 2017 (most recent test), the 72h-EC50 was 1.438 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (6) (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The evaluation of the effects was based on the average of measured exposure concentrations. The software ToxRat® Professional was used to for the determination of the ECx.

Validity criteria of the study

Cell density in Controls: 99-fold increase within 72 hours. The specific growth rate was determined at 1.53 day^-1, so greater than 0.92 day^-1.

Coefficient of Variation:

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 9.0%.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.5%.

Thus the validity criteria were respected in this study.

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mLx10⁴)

	Replicate	Nominal concentration (mg test item.L^-1) and corresponding measured concentration (mg test item.L^-1) (geometric means of measured exposure concentrations 0 - 72h for 100% test item)
		Control (0)	60 (24.39)	106 (31.74)	189 (76.24)	337 (261.45)	600 (507.94)
t=24h	1	6.0	5.6	4.0	5.2	3.2	1.2
	2	4.8	3.6	4.4	4.0	3.6	0.4
	3	4.0	4.4	4.0	4.8	5.2	0.8
	4	4.0
	5	4.4
	6	4.0
	Mean	4.5	4.5	4.1	4.7	4.0	0.8
	Std. Dev.	0.79	1.01	0.23	0.61	1.06	0.40
	% Inh.	-	0.2	3.7	-1.6	6.2	82.9
t=48h	1	19.2	17.2	21.2	13.2	4.0	1.2
	2	18.0	22.0	20.0	14.4	5.2	2.0
	3	20.8	22.4	19.2	16.4	4.4	1.6
	4	16.4
	5	18.8
	6	17.2
	Mean	18.4	20.5	20.1	14.7	4.5	1.6
	Std. Dev.	1.56	2.89	1.01	1.62	0.61	0.40
	% Inh.	-	-2.9	-2.6	6.3	39.0	68.3
t=72h	1	46.4	56.0	58.8	45.2	8.8	2.8
	2	51.2	55.2	56.4	47.6	10.8	4.0
	3	55.6	60.0	52.0	48.0	9.6	1.6
	4	40.4
	5	54.0
	6	48.4
	Mean	49.3	57.1	55.7	46.9	9.7	2.8
	Std. Dev.	5.55	2.57	3.45	1.51	1.01	1.20
	% Inh.	-	-3.3	-2.8	1.0	35.3	63.9

Table 6.1.5/2: Measured concentrations (mg/L) of the test substance in test water - final test

Nominal concentration (mg test item.L^-1)	Expected nominal concentration inD,L- DIETHYL TARTRATE (mgD,L- DIETHYL TARTRATE.L^-1)	Start (t=0h)	End (t=72h)	Relative loss to initial value (t=0h - t=72h) (%)	Geometric mean measured concentration
					mgD,L- DIETHYL TARTRATE.L^-1	%	mg test item.L^-1
Control	0	Absence	Absence	N.A.	N.A.	N.A.	N.A.
60	56.64	60.35	Absence	N.A.	23.02	40	24.39
106	100.06	102.25	Absence	N.A.	29.96	30	31.74
189	178.42	177.07	Presence	N.A.	71.97	40	76.24
337	318.13	346.59	175.76	49.29	246.81	78	261.45
600	566.40	557.61	412.33	26.05	479.50	85	507.94

N.A.: not applicable

Absence: concentrations below the LOQ (58.50 mg.L^-1) and the LOD (17.55 mg.L^-1).

Presence: concentrations below the LOQ (58.50 mg.L^-1) and above the LOD (17.50 mg.L^-).

% = Percent of expected nominal concentration in test item

Samples were centrifuged before analysis.

Where measured concentrations were below the LOQ (= presence), such concentrations were considered to be half the quantification limit, according to the study plan. Where measured concentrations were below the LOD (absence), such concentrations were considered to be half the detection limit.

Since the given geometric means measured concentrations correspond to the main constituent D,L- DIETHYL TARTRATE (purity: 94.40%), a simple calculation was performed in order to obtain values for 100% of the test item (D,L- DIETHYL TARTRATE+ impurities)

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72 -hour ErC50 for growth rate was determined to be 369.3 mg/L. The 72 -hour EC10 for growth rate was 124.8 mg/L.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item over a range of nominal test concentrations of 60, 106, 189, 337 and 600 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The concentrations of the test item were determined by chemical analyses at the start and the end of the test. Definitive test was performed based on the results of range-finding test. 

 

Chemical analyses revealed severe test item losses (due to algae metabolisation and hydrolysis). Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations. Corresponding average exposure concentrations were 24.39, 31.74, 76.24, 261.45 and 507.94 mg test item/L.

 

Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72 -hour ErC50 for growth rate was determined to be 369.3 mg/L. The 72 -hour EC10 for growth rate was 124.8 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

From 30 November 2015 to 01 December 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
iSafeRat® HA-QSAR

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O=C(OCC)C(O)C(O)C(=O)OCC
The water solubility of the substance predicted using the iSafeRat® solubility module was given as input : 1,41.10^6 mg/L

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QMRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not applicable

Remarks:

(QSAR model)

Principles of method if other than guideline:

The acute toxicity to aquatic algae was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 72-hour ErC50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 72-h ErC50 for algae and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

No additional information

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

other: Pseudokirchneriella subcapitata, Desmodesmus subspicatus, Scenedesmus quadricauda

Details on test organisms:

Following the principles of Phase Equilibrium Thermodynamics, for narcotic substances, no difference in relationship between solubility and ecotoxicity between algae (or indeed other) aquatic species is expected. Any observed differences may be attributed to lifestyle related parameters (e.g. relative differences in storage lipid content between species or shell closing in molluscs) and relative duration of study versus bodysize rather than to a specific toxic mechanism causing species differences. In this case no differences were observed in activity based toxicity for the 3 species used.

Test type:

other: QSAR method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

Not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 25 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the ErC50 values found in experimental data.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0. However it is recognized that in some cases (due to high luminosity) the pH may increase in the control and lower concentrations (which do not cause significant effect over the study period). This pH increase did not generally disqualify the study from being used in the test and validation set for non-polar chemicals.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

Not applicable.

Nominal and measured concentrations:

The QSAR is based on data from studies where analytical measurements were made on the control and all relevant test concentrations whenever possible. Any exceptions (initial concentrations measured only or ErC50 based on nominal values) were used only when sufficient justification for stability of the test was determined.

Details on test conditions:

Not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 29-221 mg/L

Details on results:

As per the iSafeRat® cascade approach (discussed in the iSafeRat® QMRF for the holistic approach, version 1.5), the subcooled liquid water solubility of a substance is strongly correlated to the ecotoxicity at different trophic levels. The toxicity to algae (72h-ErC50), which has been calculated as 81 mg/L using the predicted water solubility value (1.41.10^6 mg/L), was found to be conversative compared to the experimental result of 369.3 mg/L.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

95% CL: 29-221 mg/L.
QSAR statistical parameters are given in the QMRF and the QPRF

No additional information

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 value based on measured concentrations was determined to be 81 mg/L with 95%-Confidence Limit between 29 and 221 mg/L.

Executive summary:

A QSAR prediction (KREATiS, 2015) was performed to assess the inhibition of growth to algae of the test substance. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 201. The criterion predicted was the ErC50 (Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

 

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question (ester). This QSAR is based on validated data for a training set of 7 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was predicted using the iSafeRat solubility module at 1,41.10^6 mg/L.

The 72-h ErC50 value based on measured concentrations was determined to be 81 mg/L with 95%-Confidence Limit between 29 and 221 mg/L. This toxicity value was found to be conservative compared to the experimental study result of 369.3 mg/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) = 369.3 mg/L (95% CL: 339.0 - 403.8 mg/L) based on geometric mean measured concentrations.

72h-ErC10 (Pseudokirchneriella subcapitata) = 124.8 mg/L (95% CL: 91.8 - 152.9 mg/L) based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

369.3 mg/L

EC10 or NOEC for freshwater algae:

124.8 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, two data are available.

The first study (LPL, 2018) was assessed as a key study and was performed on the registered substance according to OECD Guideline 201 with GLP statement. In this study, the registered substance was exposed to the freshwater green algae species Pseudokirchneriella subcapitata at the nominal concentrations of 60, 106, 189, 337and 600 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The concentrations of the test substance were determined by chemical analyses at the start and the end of the test. Chemical analyses revealed severe test item losses (due to algae metabolisation and hydrolysis). Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were expressed in terms of geometric means of theexposure concentrations. Corresponding average exposure concentrations were 24.39, 31.74, 76.24, 261.45 and 507.94 mg test item/L. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72h-ErC50 and 72h-ErC10 values for growth rate were determined at 369.3 and 124.8 mg/L.

The second data (KREATiS, 2015), assessed as a supporting data, is a QSAR. This QSAR prediction (iSafeRat holistic approach v1.5) was performed on the registered substance, to assess the inhibition of growth to algae of the substance.This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 201. The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question (ester). This QSAR is based on validated data for a training set of 7 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was predicted using the iSafeRat solubility module at 1,41.10^6 mg/L. The 72-h ErC50 value based on measured concentrations was determined to be 81 mg/L (95% CL: 29 - 221 mg/L). This predicted toxicity value was found to be conservative compared to the experimental study report of 369.3 mg/L. Therefore, this QSAR result supports the key study performed on the same substance with an ErC50 value more conservative, validating the model for use with this substance.

In conclusion, according to the key experimental study (LPL, 2018), the 72h-ErC50 value on Pseudokirchneriella subcapitata is 369.3 mg/L (95% CL: 339.0 - 403.8 mg/L).