Benzoic acid, C12-15-alkyl esters

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:

Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 guideline study conducted on the registration substance in accordance with OECD guideline 201 including analytical monitoring for dose/exposure confirmation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

The objective of this study was the assessment of the effects of the test item C12-C15 alkyl benzoates (UVCB; herein referred to as test item) on the growth of the unicellular freshwater green alga Raphidocelis subcapitata (former name Pseudokirchneriella subcapitata). Due to the very low water solubility and UVCB nature of the test item, individual loading rate Water Accommodated Fractions (WAFs) were prepared according to the OECD guidance No. 23 [5]. The algae were exposed to series of nominal WAF loadings of the test item and a control (growth medium without test substance) for a period of 72 hours under static conditions in accordance with the OECD guideline 201.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch FPIL1807331

Analytical monitoring:

yes

Details on sampling:

The concentrations of test item in the water phase (algae exposure media) were measured by GC-MS chemical analysis at test initiation, after 48 hours and at the end of the test in samples taken from test solutions with and without algae. Samples (2 x about 50 – 100 mL) were taken from all five test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples, analysis and retain samples) and from the freshly prepared test cultures containing algae (2 x 5 samples). After 48 hours samples were taken from the additionally prepared test vessels containing algae and without algae (2 x 10 samples).

At the end of the growth test, samples were taken directly from the representative or pooled replicates per test concentration and controls (2 x 6 samples) and from the additional test vessels containing the test solutions without algae (2 x 5 samples).

The GC-MS analytical method was validated following SANCO/3029/99 [8] using at least two fortification levels, e.g. 1-fold LOQ and 10-fold LOQ.

Vehicle:

no

Details on test solutions:

Since the test item is a UVCB and of very low water solubility, individual loading rate Water Accommodated Fractions (WAFs) of the test item were prepared according to the OECD guidance No. 23 [5].

For each WAF loading, the required test item amount was transferred into a 2 L sterile brown glass flask (acetone washed and sterilised) that has a bottom draining port. Sterile growth medium (2 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottle and stirred slowly at 100 rpm that a small dimple was formed, avoiding bubble and foam formation for 72 hours at room temperature (about 20 °C) to achieve maximum solubility of the test item. Another flask was filled with growth medium only for the control replicates and stirred in the same way.
To separate insoluble/undissolved test item from the test medium, the bottles were allowed to stand for 24 hours. Thereafter, about 300 mL were drained off and were discarded. The next slowly released 1200 mL were used as test solution.

In a preliminary test, after the WAF preparation, measured test item concentrations were above the mean average water solubility of 2.47 μg/L at 20°C in deionized water, as determined in a separate study (INL-001/1-34). Therefore, it was concluded that micelles were formed and that the test solutions must be filtered. Accordingly, the test solutions were filtered through a 0.2 μm PES filter.

The test media preparation was conducted on a clean bench using sterile equipment.

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, Selenastraceae, Chlorophycea, Chlorophyta. (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum).
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
Cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980) [6]). Prior to testing a pre-culture will be established in modified OECD growth medium to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not Determined

Test temperature:

The culture vessels were incubated at a temperature in the range of 21 to 24 °C, controlled at ± 2°C

pH:

The solution pH was measured in one test vessel per test concentration including the control. At the end of the test, the solution pH values were measured directly in test vessels.
The test culture temperature was measured in one additional test vessel.

Dissolved oxygen:

Not Determined

Salinity:

Not Determined

Conductivity:

Not Determined

Nominal and measured concentrations:

The five loadings to be tested in the definitive test were selected on the basis of the results from the range-finding test and agreed upon with the sponsor (spacing factor of 1.8):

Control, 95.0, 171.0, 309.0, 556.0 and 1000 mg test item/L.

The concentrations of the test item were assessed by chemical analysis at test initiation, after 48 hours and at test termination of the 72-hours exposure period in samples with and without algae.

Details on test conditions:

Test vessels were 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. Test vessels were acid-washed (10% HNO3) and rinsed about six times with purified water. The vessels and silicone-sponge caps were sterilized prior to use (autoclaving).

The culture vessels were incubated at a temperature in the range of 21 to 24 °C, controlled at ± 2°C, with a light intensity (OSRAM “day light”) adjusted to 60 - 120 μE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid. The light intensity was measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in μE m-2 s-1 at level of the test media surface.

The cultures were incubated in a laboratory shaker (Incubation Shaker Multitron®, INFORS Typ HT, Switzerland) by continuous shaking on an orbital laboratory shaker (150 rpm) (INFORS, Switzerland).

Reference substance (positive control):

yes

Remarks:

Test organism sensitivity is routinely checked using 3,5-dichlorophenol twice a year. In Sept 2018 an ErC50 of 2.06 mg/L (95% CL 1.74–2.45 mg/L) is in good agreement with international ring test result of ErC50 of 3.38 ± 1.30 mg/L (mean ±SD , n=5).

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Water Accomodated Fraction (WAF)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Water Accomodated Fraction (WAF)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: Water Accomodated Fraction (WAF)

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: Water Accomodated Fraction (WAF)

Details on results:

For the examination of the inhibition of algal growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal loadings: Control, 95.0, 171.0, 309.0, 556.0 and 1000 mg test item/L.

The concentrations of the active substance in the water phase were assessed by chemical analysis using GC-MS (LOQ = 0.5 μg test item/L). Test item concentrations were determined in aqueous media samples at test initiation, after 48 hours and at test termination in samples, which were incubated with and without algae.

The test item concentration in the control was always below the LOQ at test initiation and test termination.

At test initiation, measured concentrations values of 3.10 to 6.13 μg test item/L (0.001 – 0.033 % of nominal) were found in the freshly prepared test solutions. The measured concentrations remained within the range of 2.66 to 7.83 μg test item/L (0.001 – 0.003 % of nominal) after the addition of algae. After 48 hours, measured concentrations in vessels without algae were between 0.948 to 1.98 μg test item/L, whereas it was mainly below the LOQ in the treatments with algae. At test termination, measured concentrations ranging from
The measured concentrations in test vessels without algae were comparable with the mean average water solubility value of 2.47 μg test item/L (obtained in study INL-001/1-34) which indicate the test item remained stable over 72 hours exposure. Due to the presence of the algae, measured concentrations were below the LOQ after 48 hours and 72 hours. Based on the recommendations stated in the OECD guidance No. 23 [5], the test was evaluated based on nominal loadings.

There was no concentration-dependent inhibiting effect on the growth of the freshwater green algae over the range of the tested loadings.

The 72-hour ErL50 and EyL50 for the inhibition of growth rate and yield, respectively, was above the highest nominal loading of 1000 mg test item/L and therefore also above the limit of solubility (i.e. >2.47 μg test item/L).

A NOEL above or equal to the highest nominal loading of 1000 mg test item/L and above the limit of solubility was determined for the inhibition of growth rate and yield.

Reported statistics and error estimates:

Since the test results for yield and growth rate did not show inhibition above 50 %, they were not statistically analysed to determine an EL50 and EL10 value together with 95 % confidence intervals.

The NOEL values were determined using appropriate statistical methods (Williams Multiple Sequential t-test Procedure.

The computer program ToxRat® [11] was used for statistical evaluations.

Light intensities and temperature during the test.

Date	Test duration	Light [µE s^-1m^-2]	Temperature [°C]
January 15, 2019	Test start	90.32	23.0
January 16, 2019	day 1	92.84	22.5
January 17, 2019	day 2	90.31	22.5
January 18, 2019	day 3, Test end	91.39	22.5

The pH of test solutions at test start and test end.

	Nominal WAF loading [mg test item/L]
	Control	95.0	171	309	556	1000
Test start	7.93	7.98	7.92	7.90	7.93	7.92
Test end	8.55	8.62	8.51	8.60	8.70	8.74
	8.57	8.67	8.49	8.55	8.65	8.60
	8.61	8.51	8.53	8.57	8.61	8.63
	8.57	8.58	8.51	8.57	8.62	8.65
	8.58
	8.67
	8.59
	8.61
Mean	8.59	8.59	8.51	8.57	8.64	8.65
Minimum	8.55	8.51	8.49	8.55	8.61	8.60
Maximun	8.67	8.67	8.53	8.60	8.70	8.74

Analytical results of the first rangefinder test (non-GLP).

Sample	Sampling time	Measured concentration* [µg/L water]
Range finder conc 1	Test initiation (17/09/2018)	0.591
Range finder conc 2		0.682
Range finder conc 3		0.843

*As the internal standard was found to be present in the test substances the analytical results have to be regarded as erroneous.

Analytical results of the first main test (terminated).

Sample	Sampling time	Measured concentration [µg/L water]
fresh control sample	Test initiation (06/11/2018)	<LOQ
fresh conc 1		Exceeds calibration
fresh conc 2		Exceeds calibration
fresh conc 3		Exceeds calibration
fresh conc 4		Exceeds calibration
fresh conc 5		Exceeds calibration
fresh algae conc 1		Exceeds calibration
fresh algae conc 2		Exceeds calibration
fresh algae conc 3		Exceeds calibration
fresh algae conc 4		Exceeds calibration
fresh algae conc 5		Exceeds calibration
QC 1		2.43 (97.1%)
QC 2		0.265 (106 %)

Analytical results of the 2^ndrange finding test (non-GLP).

Sample	Sampling time	Measured concentration [µg/L water]
Range finder control filt	Test initiation (11/12/2018)	<LOQ
Range finder Conc 1 filt		<LOQ
Range finder Conc 2 filt		<LOQ
Range finder Conc 3 filt		<LOQ
Range finder Kontrolle unfiltered		<LOQ
Range finder Conc 1 unfiltered		0.648
Range finder Conc 2 unfiltered		8.12
Range finder Conc 3 unfiltered		Exceeds calibration
QC 1		0.562 (93.6%)
QC 2		5.39 (89.9%)
Range finder Kontrolle filtered d3	Test termination (14/12/2018)	<LOQ
Range finder Conc 1 filtered d3		<LOQ
Range finder Conc 2 filtered d3		<LOQ
Range finder Conc 3 filtered d3		<LOQ
QC 1		0.569 (94.8%)
QC 2		5.837 (97.3%)

Note:Samples were filtered through a 0.2 µm polyethylene sulfone filter

Results of the chemical analysis of the test item at test initiation (0 h), after 48 hours and at test termination (72 h) in aqueous samples with and without algae.

Date	Nominal WAF loading [mg test item/L]	Samples including algae	Measured conc. [mg test item/L]	% of nominal
Test initiation 15/01/2019	Control	No	<LOQ
	95		0.0031	0.003
	171		0.0061	0.004
	309		0.0054	0.002
	556		0.0035	0.001
	1000		0.0051	0.001
	95	Yes	0.0027	0.003
	171		0.0078	0.005
	309		0.0053	0.002
	556		0.0035	0.001
	1000		0.0057	0.001
48 h (17/01/2019)	95	No	0.0010	0.001
	171		0.0012	0.001
	309		0.0020	0.001
	556		0.0009	0.000
	1000		0.0012	0.000
	95	Yes	<LOQ	<LOQ
	171		<LOQ	<LOQ
	309		<LOQ	<LOQ
	556		0.0006	0.000
	1000		0.0006	0.000
Test termination 18/01/2019	95	No	0.0020	0.002
	171		0.0017	0.001
	309		0.0034	0.001
	556		0.0021	0.000
	1000		<LOQ	0.000
	Control	Yes	<LOQ	<LOQ
	95		<LOQ	<LOQ
	171		<LOQ	<LOQ
	309		<LOQ	<LOQ
	556		<LOQ	<LOQ
	1000		0.0005	0.000

Inhibition of growth rate and yield compared to controls after 72 hours.

Nominal WAF loading [mg test item/L]	Growth rate		Yield
	Growth rate [1/d]	% inhibition	Yield [cells x 10^4/mL]	% inhibition
Control	1.681	-	154.3	-
95.0	1.678	0.16 (-)	153.0	0.83 (-)
171	1.707	-1.56 (-)	166.6	-7.98 (-)
309	1.663	1.06 (-)	146.3	5.21 (-)
556	1.655	1.51 (-)	142.5	7.65 (-)
1000	1.668	0.75 (-)	148.5	3.77 (-)

(+) significantly/ (-) not significantly different from controls, Williams Multiple Sequential t-test, one-sided smaller, Alpha = 0.05.

Effective loadings of the test item for the exposure ofRaphidocelis subcapitatafor 72 hours.

Parameter		EL50	NOEL
		[mg/L]
Growth rate	Value	>1000	≥1000
	Lower 95 %-cl
	Upper 95 %-cl
Yield	Value	>1000	≥1000
	Lower 95 %-cl
	Upper 95 %-cl

Microscopic observation of the algae cells at test initiation and termination of the 72 h growth test.

Date	Nominal WAF loading [mg test item/L]	Observations
Test start	Normal appearance of inoculum culture for all test concentrations.
Test end, day 3	Control	Normal appearance of algal cells.
	95.0	Normal appearance of algal cells.
	171	Normal appearance of algal cells.
	309	Normal appearance of algal cells.
	556	Normal appearance of algal cells.
	1000	Normal appearance of algal cells.

Validity criteria fulfilled:

yes

Conclusions:

A study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the toxicity of C12-C15 alkyl benzoates on the growth of the unicellular freshwater green alga Raphidocelis subcapitata. Exponentially-growing cultures of the green alga were exposed to five loadings of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

There was no concentration-dependent inhibiting effect on the growth of the freshwater green algae over the range of the tested loadings.

The 72-hour ErL50 and EyL50 for the inhibition of growth rate and yield, respectively, was above the highest nominal loading of 1000 mg test item/L and therefore also above the limit of solubility (i.e. >2.47 μg test item/L).

A NOEL above or equal to the highest nominal loading of 1000 mg test item/L and above the limit of solubility was determined for the inhibition of growth rate and yield.

Executive summary:

A study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the toxicity of C12-C15 alkyl benzoates on the growth of the unicellular freshwater green alga Raphidocelis subcapitata.

Exponentially-growing cultures of the green alga were exposed to five loadings of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test loadings were prepared with sterile growth medium under sterile conditions. Due to the very low water solubility and UVCB nature of the test item individual Water Accommodated Fractions (WAFs) of the test item was prepared according to the OECD guidance No. 23 [5]. After WAF preparation, measured test item concentrations were above the mean average water solubility of 2.47 μg test item/L at 20 °C in deionized water, as determined in a separate study (INL-001/1-34). Therefore, it was concluded that micelles were formed and that the test solutions must be filtered to avoid physical interaction of undissolved test material with algal cells that would confound interpretation of results. Accordingly, the test solutions were filtered through a 0.2 μm PES filter.

For the examination of the inhibition of algal growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal loadings: Control, 95.0, 171.0, 309.0, 556.0 and 1000 mg test item/L.

The concentrations of the active substance in the water phase were assessed by chemical analysis using GC-MS (LOQ = 0.5 μg test item/L). Test item concentrations were determined in aqueous media samples at test initiation, after 48 hours and at test termination in samples, which were incubated with and without algae.

The test item concentration in the control was always below the LOQ at test initiation and test termination.

At test initiation, measured concentrations values of 3.10 to 6.13 μg test item/L (0.001 – 0.033 % of nominal) were found in the freshly prepared test solutions. The measured concentrations remained within the range of 2.66 to 7.83 μg test item/L (0.001 – 0.003 % of nominal) after the addition of algae. After 48 hours, measured concentrations in vessels without algae were between 0.948 to 1.98 μg test item/L, whereas it was mainly below the LOQ in the treatments with algae. At test termination, measured concentrations ranging from <LOQ to 3.36 μg test item/L were found in the algal-free test solutions, whereas it was mainly below the LOQ in the treatments with algae.

The measured concentrations in test vessels without algae were comparable with the mean average water solubility value of 2.47 μg test item/L (obtained in study INL-001/1-34) which indicate the test item remained stable over 72 hours exposure. Due to the presence of the algae, measured concentrations were below the LOQ after 48 hours and 72 hours. Based on the recommendations stated in the OECD guidance No. 23 [5], the test was evaluated based on nominal loadings.

There was no concentration-dependent inhibiting effect on the growth of the freshwater green algae over the range of the tested loadings.

The 72-hour ErL50 and EyL50 for the inhibition of growth rate and yield, respectively, was above the highest nominal loading of 1000 mg test item/L and therefore also above the limit of solubility (i.e. >2.47 μg test item/L).

A NOEL above or equal to the highest nominal loading of 1000 mg test item/L and above the limit of solubility was determined for the inhibition of growth rate and yield.

Effective loadings of the test item for the exposure ofRaphidocelis subcapitatafor 72 hours.

Parameter		EL50   [mg test item/L]	NOEL  [mg test item/L]
Growth rate	Value	>1000	≥1000
	Lower 95 %-cl
	Upper 95 %-cl
Yield	Value	>1000	≥1000
	Lower 95 %-cl
	Upper 95 %-cl

Description of key information

In 2018 -2019, a study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the toxicity of C12-C15 alkyl benzoates on the growth of the unicellular freshwater green alga Raphidocelis subcapitata. Exponentially-growing cultures of the green alga were exposed to five loadings of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201. There was no concentration-dependent inhibiting effect on the growth of the freshwater green algae over the range of the tested loadings. The 72-hour ErL50 and EyL50 for the inhibition of growth rate and yield, respectively, was above the highest nominal loading of 1000 mg test item/L and therefore also above the limit of solubility (i.e. >2.47 μg test item/L). A NOEL above or equal to the highest nominal loading of 1000 mg test item/L and above the limit of solubility was determined for the inhibition of growth rate and yield.

 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

1 000 mg/L

EC10 or NOEC for freshwater algae:

1 000 mg/L

Additional information

Benzoic acid isononylester was tested in growth inhibition test with single cell green algae Desmodesmus subspicatus under static conditions according to the guidelines EU Method C.3 and OECD 201. 1 g / l benzoic acid isononylester was stirred for 18 h and undissolved test substance was then removed by filtration. All samples analysed (incl.the stock solution) were below the limit of quantification of the analytical method, i.e. < 1 mg TOC/l. Therefore, no analyses were performed on the test concentrations after 72 h. No negative effects on growth rate were recorded, thus the NOEC growth rate is > = the limit of solubility of test item under test conditions and the EC50 growth rate is > the limit of solubility (limit of detection of TOC analysis = 1 mg/L). In the concentration range tested, the test item had a positive effect on the growth of the algae.