Acetylcysteine

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:

2018-04-10 to 2018-05-17

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:

corrected July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

August 24, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. Additional samples of the control without algae were taken at each sampling without any sample treatment.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.7 mg test item into 1007 mL test water by intense stirring for 35 minutes. The pH of the test medium was adjusted with 1 M NaOH from 4.7 to 8.1. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Controls: In the control, test water was used without addition of the test item
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

- Source: algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany
- Breeding conditions: algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.3 to 22.9 °C

pH:

8.1 at test start and 9.3 to 9.7 at test end

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: 100, 32, 10 and 1.0 mg/L (spacing factor 3.16)
Measured: 100, 32, 10 and 1.0 mg/L (100 % of nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Size, fill volume: 50 mL volume with approximately 50 mL of test medium
- Aeration: No
- Initial cells density: approx. 5000 cells per mL test medium
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM
- Dilution water: test medium was prepared with deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 5765 lux (range: 5440 to 6000 lux)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: spectrophotometric measurement

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes, non-GLP
- Test concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a blank control
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to the highest concentration tested.
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: EyC50 (yield) = 0.402 mg/L, ErC50 (growth rate) = 0.963 mg/L, EC50 (biomass) = 0.414 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 could not be calculated by Probit analysis due to the lack of effects and were therefore determined directly from the raw data. For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Table 1: Yield y and percentage inhibition of y during the test period

Nominal concentration	Yield and percentage inhibition ofy during the test period
	0 - 24hours		0 - 48hours		0 - 72hours
[mg test item/L]	y	%	y	%	y	%
Control	2.421	-	19.394	-	102.093	-
1	2.457	-1.5	19.542	-0.8	98.725	3.3
3.2	2.892	-19.4	19.099	1.5	104.162	-2.0
10	2.675	-10.5	18.877	2.7	95.074	6.9
32	2.602	-7.5	18.507	4.6	100.673	1.4
100	2.964	-22.4	18.655	3.8	101.160	0.9

 negative values in '% inhibition' indicate an increase in growth relative to that of the control mean value

*significantly different from the control (tested with Williams t-test (24-72h),a= 0.05,one sided)

 

Table 2: Growth rates µ and % inhibition of µ

Nominal concentration	Growth rates µ [1/day] and % inhibition of µ
	0-24 hours		0–48hours		0-72 hours
[mg/L]	µ	%	µ	%	µ	%
Control	1.764	-	1.839	-	1.774	-
1	1.777	-0.7	1.845	-0.3	1.763	0.6
3.2	1.913	-8.4	1.831	0.4	1.779	-0.3
10	1.848	-4.8	1.827	0.7	1.750	1.3
32	1.825	-3.5	1.818	1.1	1.770	0.2
100	1.933	-9.6	1.822	0.9	1.771	0.2

negative values in '% inhibition' indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Williams t-test (24-72h),a= 0.05,one sided)

 

Table 3: Analytical results

Sample Description	% of	RSD	n
[mg test item/L]	nominal^1	[%]
Control	n.a.	n.a.	2
100	100	5	2

¹ mean value of all measured samples per treatment group RSD: relative standard deviation per treatment group n: number of analysed samples n.a.: not applicable

Validity criteria fulfilled:

yes

Conclusions:

In a Algal Growth Inhibition Test according to OECD TG 201 and EU Method C.3 the 72-hour EC50, EC10 and NOEC of the test item for yield and growth rate, respectively, were determined to be > 100 mg/L.

Executive summary:

A 72-hour Algal Growth Inhibition Test according to OECD TG 201 and EU ethod C.3 was conducted in compliance with GLP principles to determined the inhibitory effect of the test item to the freshwater green algae Raphidocelis subcapitata . For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The study encompassed 6 treatment groups (100, 32, 10, 3.2 and 1.0 mg test item/L) with three replicates per test concentration and six replicates for the blank control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities by spectrophotometrical measurement. The quantification of the test item in the test sample was performed via measurement of Total Organic Carbon (TOC). In the analytical part the initial concentration and the maintenance of the exposure concentration during the test were verified for the highest test concentration of 100 mg test item/L. Since the concentrations of the test item were within ± 20 % of the nominal concentrations during the test, results refer to nominal values. The 72-hour NOEC was determined to be at least 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 were higher than 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to nominal 100 mg test item/L. All validity criteria of the guidelines were fulfilled.

Description of key information

In a Algal Growth Inhibition Test according to OECD TG 201 and EU Method C.3 the 72-hour EC50 and EC10 of the test item for yield and growth rate, respectively, were determined to be > 100 mg/L. The 72-hour NOEC for yield and growth rate were determined to be > 100 mg/L (reference 6.1.5 -1).

Additional information

To determine the inhibitory effect of the test item to the freshwater green algae Raphidocelis subcapitata a 72-hour Algal Growth Inhibition Test according to OECD TG 201 and EU Method C.3 was conducted in compliance with GLP principles. Exponentially growing green algal were exposed to 100, 32, 10, 3.2 and 1.0 mg test item/L with three replicates per test concentration and six replicates for the blank control. The cell densities of test samples was measured spectrophotometrically after 24, 48 and 72 hours of exposure. The test item concentrations in the test solutions were confirmed by measurement of the Total Organic Carbon (TOC) to be within ± 20 % of the nominal concentrations and hence, test results refer to nominal values. The 72-hour NOEC was determined to be > 100 mg test item/L. The 72-hour EC50 and EC10 were found to be higher than 100 mg test item/L. All validity criteria of the guidelines were fulfilled (reference 6.1.5 -1).