Ethyl 3,4-dihydroxybenzoate

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:

17 Juni 2016 to 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 3,4-dihydroxybenzoate

EC Number:

223-529-0

EC Name:

Ethyl 3,4-dihydroxybenzoate

Cas Number:

3943-89-3

Molecular formula:

C9H10O4

IUPAC Name:

ethyl 3,4-dihydroxybenzoate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Pre test/range finding
- Concentrations: 0.1 and 10 mg/L at 0, 24 and 72 hours
- Sampling method: single 2.4 mL samples (pooled at 72 hours)
- Sample storage conditions before analysis: stored in freezer
Main study
- Concentrations: 0, 1.0, 1.8, 3.2, 5.6 and 10 mg/L at 0, 24 and 72 hours
- at 1,0 mg/L in a replicate without algae at 0, 24 and 72 hours
- Sampling method: single 2.4 mL samples (pooled at 72 hours)
- Sample storage conditions before analysis: stored in freezer

Reserve samples were taken but not used.

Test solutions

Vehicle:

no

Details on test solutions:

Starting with a concentration of 100 mg/L. Magnetic stirring for 1 hour (comb. limit/range-finding) or 1.25 hour (final test) resulted in a completely dissolved solution. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/L solution in test medium. All final test solutions were clear and colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in house culture
- Method of cultivation: 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.
- Inoculum medium: M1

Acclimatisation:
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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C

pH:

8.0-8.3

Nominal and measured concentrations:

nominal: 0, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
measured (72h TWA): ND, 0.035, 0.17, 0.60, 1.9 and 6.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL
- Type: closed
- Material: all-glass, containing 50 mL of test solution
- Aeration: none
- Initial cells density: 1E04 cells/mL
- Control end cells density: 151.7E04 cells/mL
- No. of vessels per concentration (replicates): 3 per concentration
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, M1 (according to the guideline)

TEST MEDIUM / WATER PARAMETERS: M2 according to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:continues
- Light intensity and quality: TLD-lamps with a light intensity within the range of 83 to 89 µE.m-2.s-1

EFFECT PARAMETERS MEASURED: growth rate at 24, 48 and 72 hr; biomass at 0, 24, 48 and 72 hr
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Range finding study: 0, 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
growth rate: 1.664, 1.694, 1.501, 0.266 and 0.513 at 0, 0.1, 1.0, 10 and 100 mg/L
yield 146.8, 160.5, 89.3, 1.2 and 3.7 cells/mL at 0, 0.1, 1.0, 10 and 100 mg/L

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CV 2.3 to 2.5 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% CV 0.88 to 1.1 mg/L

Details on results:

validity criteria fulfilled:
1. In the control, cell density increased by an average factor of 152 within the exposure period
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 11.2%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.1%

Results with reference substance (positive control):

Potassium dichromate: 72h-ERC50 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L.

Reported statistics and error estimates:

EC50 values calculated by Probit analyses
Growth rate and yield inhibition NOEC by Williams Multiple Sequential t-test

Any other information on results incl. tables

Percentage inhibition of growth rate (total test period) during the final test

CH02700 concentration TWA (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.672	0.0352	6
0.035	1.685	0.0427	3	-0.8
0.17	1.713	0.0280	3	-2.5
0.60	1.604	0.0220	3	4.1*^/#
1.9	1.010	0.0370	3	39.6*
6.1	0.224	0.0544	3	86.6*

* - effect was statistically significant

# - effect was biologically insignificant

 

Percentage inhibition of yield during the final test

CH02700 concentration TWA (mg/L)	Mean	Std. Dev.	n	%Decrease
Control	150.7	16.26	6
0.035	156.8	19.52	3	-4.0
0.17	170.2	14.29	3	-12.9
0.60	122.3	8.28	3	18.9*
1.9	19.8	2.37	3	86.9*
6.1	1.0	0.33	3	99.4*

* - effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72h-ERC50 was 2.4 mg/L

Executive summary:

In a test according to OECD 201, algae were exposed to 0, 0.10, 0.32, 1.0, 3.2 and 10 mg/L (measured 72h TWA: ND, 0.035, 0.17, 0.60, 1.9 and 6.1 mg/L). A significant decrease of biomass was observed at 0.17 mg/L and above, while growth rate was significantly decreased (and biologically relevant) at 0.60 mg/L. 72h-ERC50 was 2.4 mg/L based on TWA measured concentrations..