Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

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:

15 August 2011 to 15 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

435-360-1

EC Name:

-

Cas Number:

144447-11-0

Molecular formula:

C20H22O5

IUPAC Name:

4-methoxyphenyl 4-[4-(oxiran-2-yl)butoxy]benzoate

Details on test material:

Identification: Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester
Chemical name: 4-methoxyphenyl-4(5,6-epoxyhexyloxy)benzoate
Molecular formula: C20H22O5
Molecular weight: 342.39
CAS Number: 144447-11-0
Description: White to off-white powder
Batch: 1001501002
Purity: 98.2%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 February 2013

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Stability in water: Not indicated
Solubility in water: 0.586 mg/L (NOTOX project 497269)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing undissolved residue was kept for possible analysis.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml
Storage: Samples were stored in a freezer until analysis.

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 demonstrated by running a test vessel at the highest substance 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.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester tested was a white to off-white powder with a purity of 98.2% and not completely soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 2-day period of magnetic stirring to ensure maximum dissolution. The obtained mixture containing undissolved material was filtered through a 0.45 µm membrane filter (Whatman, RC55). The undiluted filtrate was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

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 10^4 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Source: In-house laboratory culture.

Reason for selection: This system is an unicellular algal species sensitive to toxic substances 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-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation 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:

no

Total exposure duration:

72 h

Remarks on exposure duration:

-

Post exposure observation period:

none

Test conditions

Hardness:

0.24 mmol(24 mg CaCO3/L)

Test temperature:

21.7 and 23.0°C

pH:

7.8-8.3

Dissolved oxygen:

not measured

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations:

Solutions containing 0.1, 3.2, 10, 32 and 100% of the filtrate prepared at a loading rate of 100 mg/l.

Measured concentrations:

The actual concentrations were measured in samples taken from the solutions containing 10, 32 and 100% of the filtrate. At the start of the test, the actual test concentrations were 0.034, 0.12 and 0.37 mg/l, respectively. The measured concentrations were below the limit of detection of the analytical method, i.e. 0.013 mg/l, at the end of the test.

The calculated Time Weight Average (TWA) concentrations were 0.009, 0.0.13 and 0.091 mg/l in solutions containing 10, 32 and 100% of the filtrate prepared at a loading rate of 100 mg/l.

Details on test conditions:

FINAL STUDY:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1598000 cells/ml
Replicates:
6 replicates of the control and the highest concentration
3 replicates of each lower test concentration
1 replicate of each concentration without algae

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 68 to 74 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.02-0.14 mg/ll

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.091 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.01-0.07 mg/l

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.02-0.32 mg/l

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Details on results:

Growth rates were in the range of the controls in all groups but the highest one during the 72-hour test period. In the highest group the growth rate was reduced by 13%. This effect was also statistically significant (Dunnett’s test, α = 0.05).

Yield was significantly reduced at the highest concentration tested (Dunnett’s test, α = 0.05).

The observed increase of growth and stimulation of yield at the TWA concentration of 0.009 mg/l were statistically significant but considered biologically not relevant.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/l with a 95% confidence interval ranging from 1.1 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 0.34 to 0.78 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.
- Results with reference substance valid? yes
- EC50: was 1.5 mg/l with a 95% confidence interval ranging from 1.1 to 2.1 mg/l; The EC50 for yield inhibition (EYC50: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 0.34 to 0.78 mg/l.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Dunnett’s test, SAS Enterprise Guide v.4). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test substance.

In case concentrations measured are below the limit of detection, the final exposure concentration(s) will be taken as a factor of 2 below the limit. This procedure is based on the OECD “Guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment”.

No EC50-value for growth reduction could be calculated (EC50 > maximum concentration tested).

Any other information on results incl. tables

Mean cell densities (x 10⁴cells/ml) during the final test

Test substance^1 % of WSF^2	Exposure time (hours)
	0	24	48	72
control	1.0	11.2	49.3	159.8
1.0	1.0	11.9	51.1	158.9
3.2	1.0	12.2	54.5	180.0
10 (0.009)	1.0	11.5	55.8	196.2
32 (0.013)	1.0	10.4	42.1	143.3
100 (0.091)	1.0	6.9	20.4	81.4

1.Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

2. Water Soluble Fraction prepared at 100 mg/l

() – between bracket TWA concentrations are given [mg/l]

Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test substance^1 % of WSF^2	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.07043		158.77
1.0	0.07039	0.0	157.93	0.5
3.2	0.07208	-2.3	179.04	-12.8
10 (0.009)	0.07329	-4.1	195.21	-23.0
32 (0.013)	0.06894	2.1	142.35	10.3
100 (0.091)	0.06098	13.4	80.37	49.4

1.Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

2. Water Soluble Fraction prepared at 100 mg/l

() – between bracket TWA concentrations are given [mg/l]

Percentage reduction of growth rate at different time intervals during the final test

Test substance^1 % of WSF^2	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
control	0.10049		0.06192		0.04887
1.0	0.10309	-2.6	0.06077	1.9	0.04732	3.2
3.2	0.10415	-3.6	0.06237	-0.7	0.04971	-1.7
10 (0.009)	0.10176	-1.3	0.06576	-6.2	0.05236	-7.1
32 (0.013)	0.09769	2.8	0.05815	6.1	0.05098	-4.3
100 (0.091)	0.08030	20.1	0.04467	27.9	0.05796	-18.6

1.Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

2. Water Soluble Fraction prepared at 100 mg/l

() – between bracket TWA concentrations are given [mg/l]

pH levels recorded during the final test

Test substance^1 % of WSF^2	Exposure time (hours)
	0	72
control	8.2	8.0
1.0	8.2	7.9
3.2	8.1	7.9
10 (0.009)	8.1	7.9
32 (0.013)	8.2	7.9
100 (0.091)	8.3	7.8

1.Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

2. Water Soluble Fraction prepared at 100 mg/l

() – between bracket TWA concentrations are given [mg/l] 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 0.091 mg/l, i.e. an initially prepared loading rate of 100 mg/l.

The EC50 for growth rate reduction (ERC50: 0-72h) was beyond the range tested, i.e. exceeded a TWA concentration of 0.091 mg/l (initially prepared at a loading rate of 100 mg/l).

The EC50 for yield inhibition (EYC50: 0-72h) was 0.09 mg/l with a 95 % confidence interval ranging from 0.02 to 0.32 mg/l based on TWA concentrations.

The EC10 for growth reduction (ERC10: 0-72h) was 0.05 mg/l with a 95% confidence interval ranging from 0.02 to 0.14 mg/l based on TWA concentrations.

The EC10 for yield inhibition (EYC10: 0-72h) was 0.02 mg/l with a 95% confidence interval ranging from 0.01 to 0.07 mg/l based on TWA concentrations.