Dioctyl maleate, branched

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2017 - 16 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Substance Name: Bernel Ester DCM
CAS Number: 85566-63-8
Purity: 92.78%
Receipt Date: 4 September 2017
Storage on Receipt: Room temperature (15 – 30°)

Analytical monitoring:

yes

Details on sampling:

At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control and each test concentration for chemical analysis.
At 72 hours, ca 20 mL samples were taken for chemical analysis from the pooled old test media from the control and each test concentration.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

no

Details on test solutions:

Test Procedures
Based on the low solubility of the compound, which was stated as “insoluble in water” on the MSDS (material safety data sheet), a saturated solution preparation was considered most suitable (OECD 23).

Test organisms (species):

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

Details on test organisms:

Test Organism
The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.
Prior to testing, duplicate starter cultures were prepared and incubated under test conditions (as detailed in Appendix 2) to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 104 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

CaCl2.2H2O 18 mg/l

Test temperature:

23.3 – 23.5 °C

pH:

8.06-9.00

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Range-finding test: 1.0, 10 & 100% saturated solution (nominal)
Definitive test: 1.0, 3.2, 10, 32 &100% saturated solution (nominal)
Definitive test:

Details on test conditions:

Test Vessel Preparation:
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added. Sterile foam bungs were used to cover the top of the vessels.
Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 × 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis.

- Test Vessel Sampling:
At approximately 24-hour intervals after the start of the incubation period, pre-determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48 and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

- Environmental Conditions:
All flasks were loosely-capped and incubated in a temperature controlled laboratory under a light bank. The vessels were placed on an orbital shaker (ca 100 rpm) under conditions of constant light (4440 to 8880 Lux), using florescent tube lights, emitting
light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test.
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital thermometer.

- Data Interpretation:
The algal cell concentration data were evaluated using three approaches:

(a) Comparison of average specific growth rates (µ)
The average specific growth rate (µ) for test algae in each test vessel was calculated using the equation:

µ = (Loge Nm - Loge N0) / (tn - t1)

where:
N0 = initial measured cell concentration at time t0 (cells/mL)
N1 = measured cell concentration at time t1 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
N2 = measured cell concentration at time t2 (cells/mL)
t1 = time of first measurement after the beginning of the test (h)
t2 = time of second measurement after the beginning of the test (h)
tn = time of nth measurement after the beginning of the test (h)

The percentage inhibition (IA%) of cell growth was calculated using the following
equation:

I (%) = ((Ac - As) / Ac) x 100

where:
Ac = mean values of A or µ determined for the control treatment
As = mean values for A or µ determined for each treatment concentration (As)
Section-by-section percentage inhibition in growth rate and section-by-section growth
rates for control vessels are also reported.

(b) Final Yield (Y)
Yield (in terms of biomass) was determined using the following formula:
Yield = final time point biomass (cells/mL) – initial (inoculum) biomass (cells/mL)
Yields were calculated for each test vessel and a mean yield per treatment was
determined.

- Statistical Analysis:
Statistical analysis was not performed as no toxicity was observed during the test.

- Chemical Analysis:
Analytical chemistry was performed during the definitive phase, however, the analytical batch run at 0 hours failed to meet acceptance criteria. Therefore, a decision was made alongside the client to not report the analytical data, as over the course of
the study analytical method criteria was not met, this was considered to be due to issues with the compound and method (discussed further in Test Substance Properties and Related Issues section of the report). As analytical work was not reliable results were based on nominal loading rate concentrations (mg/L LR WAF).

- Mathematical Rounding Statement:
For presentation purposes, some numerical data may have been rounded, therefore manual recalculation may result in slightly different values to those presented in the results section.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.251 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.251 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.152 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.009 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Range-finder test
Key results from the range-finding test are summarised in Table 1.
At the start of the test, the control and test concentration were observed to be colourless solutions. At 72 hours, the control and 1.0% saturated solution test concentration were observed to be green, homogenous hazy dispersions of algal cells, whereas the 10 and 100% saturated solution test concentrations were observed to be pale green, homogenous hazy dispersions of algal cells.
Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that the 72-hour EC50 values for yield and specific growth rate were likely to be between 10 and 100% saturated solution test concentrations, based on nominal test substance concentration.

Definitive Test
Chemical Analysis
The results of the chemical analysis are presented in Table 2.
Example chromatograms of test samples, standard solution and a typical calibration line are presented in Appendix 3.
The limit of quantification (LOQ) for Bernel Ester DCM in EC medium using this method was 0.005 mg/L.
Analysis of the test media samples was conducted on fresh media at 0 hours, analysis of the corresponding old media was conducted at 72 hours.
Analysis of the test samples at 0 and 72 hours are summarised in the table below.
Chemical analysis showed Bernel Ester DCM declined in concentration over the 72-hour test period, therefore, results were based on geometric mean measured concentrations. For the results at 72 hours that were below the LOQ (limit of quantification) a value of half the LOQ was used to calculate the mean values based on guidance from OECD 23. The geometric mean measured concentrations were calculated to be 0.005, 0.009, 0.056 and 0.251 mg/L. A geometric mean value was not calculated for the 1.0% saturated solution test group as both 0 and 72 hour results were below the LOQ, this was not considered to impact on study integrity as this level was below the NOEC (no observed effect concentration).

Environmental Conditions
The results of the environmental conditions and pH determined during the test are presented in Table 3.
Temperatures remained within 21 - 24ºC, and were maintained within ± 2ºC range established for the test, temperature range during the test was 23.3 – 23.5°C.
The light intensity was measured at the start and end of the test; 5675 Lux at the start and 5948 Lux at the end of the test (mean of four readings carried out at each time point).
The pH of the test solutions ranged from 7.74 to 7.85 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata. The pH in the controls increased by a maximum of 1.07 units.

Test Media Descriptions
The freshly prepared test media at 0 hours appeared as colourless solutions.
The appearance of the test media following the 72-hour exposure period are summarised in the table on the following page.

Growth Test
Individual replicate data for cell density and mean treatment cell density are presented in Table 4. Results of the growth test, as specific growth rate and yield are presented in Table 5 to Table 8.
Growth curves of Pseudokirchneriella subcapitata exposure to Bernel Ester DCM are presented in Figure 1.
The 72-hour yield (EyCx), and growth rate (ErCx) toxicity values, with corresponding no observed effect concentrations (NOEC) are presented in the table below.
Based on geometric mean measured concentrations, the 72-hour EyC50 and ErC50 values were calculated to be 0.152 and >0.251 mg/L, respectively.
The corresponding NOEC values for yield and specific growth rate after 72 hours were 0.009 and 0.251 mg/L respectively.

Validity Criteria
The validity criteria specified in the OECD 201 Test Guideline (Reference 1) are;
i. To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72-hour test period. In this test, the cell density increase (as a surrogate for biomass) was approximately 88 fold over the 72 hours.
ii. The mean coefficient of variation for the control replicate sectional (daily) specific growth rates must not exceed 35%. This was determined to be 21.85% in this test.
iii. The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7%. This was 3.61% in this test.
All validity criteria were met therefore the test was considered valid.

Definitive test - Chemical analysis

Nominal Concentration (% saturated solution)	Measured Concentration (mg/L)
	0 hours (New)	72 hours (Old)
Control	-	-
1	<LOQ	<LOQ
3.2	0.0106	<LOQ
10	0.0315	<LOQ
32	0.134	0.0238
100	0.404	0.156
-	None found
LOQ = Limit of quantification (0.005 mg/L)

Definitive test - test media descriptions

Nominal Concentrations (% saturated solution)	Colour of inoculated test medium	State of test medium
Control	green	homogenous hazy dispersion of algal cells
1
3.2
10
32
100	pale green

Definitive test - Dose descriptors

Parameter		Toxicity Values (mg/L)		Statistical Test
		72 hours	Confidence limits (LCL-UCL)
Yield	EyC10	0.00423	NC – 0.0258	Linear Interpolation (ICPIN)
	EyC20	0.0182	NC – 0.0383
	EyC50	0.152	NC – 0.351
	NOEC	0.009	NC	Bonferoni Adj t Test
Growth Rate	ErC10	0.0453	0.00754 – 0.120	Linear Interpolation (ICPIN)
	ErC20	>0.251	NC
	ErC50	>0.251	NC
	NOEC	0.251	NC	Derived empirically
NC	Not calculated
LCL	Lower confidence limit
UCL	Upper confidence limit

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met therefore the test was considered valid.

Conclusions:

The effects of Bernel Ester DCM on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

Chemical analysis showed Bernel Ester DCM declined in concentration over the 72-hour test period, therefore, results were based on geometric mean measured concentrations. These were calculated to be 0.005, 0.009, 0.056 and 0.251 mg/L. A geometric mean value was not calculated for the 1.0% saturated solution test group as both 0 and 72 hour results were below the LOQ, this was not considered to impact on study integrity as this level was below the NOEC values.

Based on geometric mean measured concentrations, the 72-hour EyC50 and ErC50 values were calculated to be 0.152 and >0.251 mg/L, respectively.
The corresponding NOEC values for yield and specific growth rate after 72 hours were 0.009 and 0.251 mg/L, respectively.
All validity criteria were met therefore the test was considered valid.

Executive summary:

The objective of the study is to determine the effects of the test substance against algal growth by exposing the green alga,Pseudokirchneriella subcapitata,at the exponential growth phase to the test substanceduring a 72-hour test period.

The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201.

Based on the results of a range-finding test, for which the key results only have been reported, a definitivetest was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solution. Six replicate vessels were prepared for the control, three replicate test vessels were prepared for the test concentrations.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with
1 × 10⁴ algae cells/mL and incubated at 21 – 24°C (under 4440 – 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Measured test concentrations were determined from samples of test solution from treatment and control groups at the beginning of the test (new media) and the test end (old media).

Analysis of the test samples at 0 and 72 hours are summarised in the table below.

Nominal Concentration (% saturated solution)		Measured Concentration (mg/L)
		0 hours (New)	72 hours (Old)
Control		-	-
1.0		<LOQ	<LOQ
3.2		0.0106	<LOQ
10		0.0315	<LOQ
32		0.134	0.0238
100		0.404	0.156
-	None found
LOQ = Limit of quantification (0.005 mg/L)

Chemical analysis showed Bernel Ester DCM declined in concentration over the 72-hour test period, therefore, results were based on geometric mean measured concentrations. For the results at 72 hours that were below the LOQ (limit of quantification) a value of half the LOQ was used to calculate the mean values based on guidance from OECD 23. The geometric mean measured concentrations were calculated to be 0.005, 0.009, 0.056 and 0.251 mg/L. A geometric mean value was not calculated for the 1.0% saturated solution test group as both 0 and 72 hour results were below the LOQ, this was not considered to impact on study integrity as this level was below the NOEC (no observed effect concentration).

The 72-hour yield (E_yC_x), and growth rate (E_rC_x) toxicity values, with corresponding no observed effect concentrations (NOEC) are presented in the table below. 

Parameter		Toxicity Values (mg/L)		Statistical Test
		72 hours	Confidence limits (LCL-UCL)
Yield	EyC10	0.00423	NC – 0.0258	Linear Interpolation (ICPIN)
	EyC20	0.0182	NC – 0.0383
	EyC50	0.152	NC – 0.351
	NOEC	0.009	NC	Bonferoni Adj t Test
Growth Rate	ErC10	0.0453	0.00754 – 0.120	Linear Interpolation (ICPIN)
	ErC20	>0.251	NC
	ErC50	>0.251	NC
	NOEC	0.251	NC	Derived empirically
NC	Not calculated
LCL	Lower confidence limit
UCL	Upper confidence limit

 Based on geometric mean measured concentrations, the 72-hourE_yC₅₀and E_rC₅₀values were calculated to be 0.152 and >0.251 mg/L, respectively.

The corresponding NOEC values for yield and specific growth rate after 72 hours were 0.009 and 0.251 mg/L respectively.

All validity criteria were met therefore the test was considered valid.

Description of key information

The key study was conducted according to internationally recognised testing guidelines and with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.251 mg/L