3,4-dihydroxybenzonitrile

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:

2017

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)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 3.6 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
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 checked by running a test vessel at the limit 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 samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 515444.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of CH02906 tested was a white to brownish powder with a purity of 99.9% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a concentration of 100 mg/L for the combined limit/range-finding test and 10 mg/L for the final test applying a stirring period of 10-15 minutes to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the 100 or the 10 mg/L concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

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 (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation:
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).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

7.7-8.3
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).

Nominal and measured concentrations:

0.10, 0.32, 1.0, 3.2 and 10 mg/L.
At the start of the test, the measured concentrations were in agreement with nominal (87-99%). The three highest test concentrations remained relatively stable during the test period and ranged between 80 and 85% of nominal at the end of the test. The two lowest concentrations decreased and ranged between 26 and 55% of nominal at the end of the test. Based on these results, the time weighted averge (TWA) concentrations were calculated and used for the determination of the effect parameters.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/ml
- Control end cells density: 358 * 10^4 cells/ml
- No. of organisms per vessel: 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 extra replicate of the control and each test concentration for sampling at 24 hours;
1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24h
- Light intensity and quality: TLD-lamps with a light intensity within the range of 77 to 84 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (June 2017)

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Biologically relevant NOEC

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

whereas the growth rate of algae exposed to 0.24 mg/L and higher were increasingly inhibited. Statistically significant inhibition of growth rate was found at test concentrations of 0.24 mg/L and higher. However, biologically relevant inhibition was seen only at concentrations of 0.89 mg/L and higher.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 19%).
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2%).
Inhibition of yield increased with increasing concentration of CH02906 from 0.046 mg/L upwards resulting in >99% inhibition at and above 2.7 mg/L. Statistically significant inhibition of yield was found at test concentrations of 0.24 mg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.89 mg/L when compared to the control.

Results with reference substance (positive control):

The 72h-NOEC for growth rate inhibition was 0.046 mg/L when based on statistical analysis and 0.24 mg/L when based on biological relevance.

The 72h-EC10 for growth rate inhibition (ERC10) was 0.29 mg/L with a 95% confidence interval ranging from 0.27 to 0.32 mg/L.

The 72h-EC50 for growth rate inhibition (ERC50) was 1.4 mg/L with a 95% confidence interval ranging from 1.3 to 1.5 mg/L.

The 72h-NOEC for yield inhibition was 0.046 mg/L when based on both statistical analysis and biological relevance.

The 72h-EC10 for yield inhibition (EYC10) was 0.14 mg/L with a 95% confidence interval ranging from 0.12 to 0.16 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 0.38 mg/L with a 95% confidence interval ranging from 0.35 to 0.41 mg/L.

Reported statistics and error estimates:

See table 1

Table 1          
Effect Parameters

	Parameter (mg/L)	NOEC (stat)*	NOEC (biol)^#	EC10	EC20	EC50
Growth rate	Value	0.046	0.24	0.29	0.50	1.4
	lower 95%-cl			0.27	0.47	1.3
	upper 95%-cl			0.32	0.53	1.5
Yield	Value	0.046	0.046	0.14	0.19	0.38
	lower95%-cl			0.12	0.17	0.35
	upper95%-cl			0.16	0.22	0.41

*Statistically determined NOEC;^#Biologically relevant NOEC; cl: confidence limits

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, CH02906 inhibited both growth rate and yield of this fresh water algae species at TWA concentrations of 0.24 mg/L and higher.
The 72h-NOEC for growth rate inhibition was 0.046 mg/L when based on statistical analysis and 0.24 mg/L when based on biological relevance.
The 72h-EC10 for growth rate inhibition (ERC10) was 0.29 mg/L with a 95% confidence interval ranging from 0.27 to 0.32 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.4 mg/L with a 95% confidence interval ranging from 1.3 to 1.5 mg/L.
The 72h-NOEC for yield inhibition was 0.046 mg/L when based on both statistical analysis and biological relevance.
The 72h-EC10 for yield inhibition (EYC10) was 0.14 mg/L with a 95% confidence interval ranging from 0.12 to 0.16 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.38 mg/L with a 95% confidence interval ranging from 0.35 to 0.41 mg/L.

Executive summary:

The objectiveofthe study was to evaluate CH02906 for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀ and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201 (2006; Annex 5 corrected 28 July 2011).

The batch of CH02906 tested was a white to brownish powder with a purity of 99.9% and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed tonominal CH02906 concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The initial algal cell density was 10⁴cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the measured concentrations were in agreement with nominal (87-99%). The three highest test concentrations remained relatively stable during the test period and ranged between 80 and 85% of nominal at the end of the test. The two lowest concentrations decreased and ranged between 26 and 55% of nominal at the end of the test. Based on these results, the time weighted average (TWA) concentrations were calculated to be 0.046, 0.24, 0.89, 2.7 and 8.9 mg/L and used for the determination of the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

	Parameter (mg/L)	NOEC (stat)*	NOEC (biol)^#	EC10	EC20	EC50
Growth rate	Value	0.046	0.24	0.29	0.50	1.4
	lower 95%-cl			0.27	0.47	1.3
	upper 95%-cl			0.32	0.53	1.5
Yield	Value	0.046	0.046	0.14	0.19	0.38
	lower95%-cl			0.12	0.17	0.35
	upper95%-cl			0.16	0.22	0.41

*Statistically determined NOEC;^#Biologically relevant NOEC (>10% effect); cl: confidence limits

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.24 mg/L