N-acetylsulphanilyl chloride

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The stock solution (100 mg/L) was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept in ultrasonic bath for 20 mins. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5x10(3) cells /ml
- Method of cultivation: No data available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Test temperature:

23±2°C

pH:

without adjustment
the sample at concentration:
10 mg/l: pH = 7.7 (at the start of test) changed to 7.4 (at the end of the test)
17 mg/l: pH = 7.5 (at the start of test) changed to 7.3 (at the end of the test)
29 mg/l: pH = 7.4 (at the start of test) changed to 7.2 (at the end of the test)
49 mg/l: pH = 6.9 (at the start of test) changed to 6.8 (at the end of the test)
84 mg/l: pH = 6.4 (at the start of test) changed to 4.7 (at the end of the test)

control: pH = 8.0 changed to pH = 7.5 during the test

Nominal and measured concentrations:

Nominal test chemical conc. used for the study were 0, 10, 17, 29, 49 and 84 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15 ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

70.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 65.2 - 76.8 mg/l

Results with reference substance (positive control):

- Results with reference substance valid
- EC50: 0.77 mg/L (24 hours)

Reported statistics and error estimates:

EC50 was calculated using non linear regression by the software Prism 4.0

In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.8%.

Result of the definitive test:

Sample no.	Sample Information	Conc. (mg/l)	µ (d-1)	I%	EC50
					mg/l	95% C. I. (mg/l)
Control	OECD medium	0	2.01	-
Test chemical	Test chemical	10 17 29 49 84	1.86 1.85 1.84 1.84 0.27	1.1 1.6 2.1 2.1 85.6	70.8	65.2 to 76.8

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical, the 72hr median effective concentration (ErC50) value was determined to be 70.8 mg/l (95% CL: 65.2 - 76.8 mg/l) (nominal concentration).

Executive summary:

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (100 mg/L) was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept in ultrasonic bath for 20 mins. Test solutions  of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 10, 17, 29, 49 and 84 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.8% i.e, not exceeded 7% and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 70.8 mg/l (95 % CI 65.2 - 76.8 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2018). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (100 mg/L) was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept in ultrasonic bath for 20 mins. Test solutions  of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 10, 17, 29, 49 and 84 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.8% i.e, not exceeded 7% and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 70.8 mg/l (95 % CI 65.2 - 76.8 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

70.8 mg/L

Additional information

Various experimental studies and predicted data of the target chemical and supporting weight of evidence study of its structurally similar read across substance were reviewed for the toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental key study from study report (2018), toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (100 mg/L) was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept in ultrasonic bath for 20 mins. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 10, 17, 29, 49 and 84 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.8% i.e, not exceeded 7% and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 70.8 mg/l (95 % CI 65.2 - 76.8 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris (Experimental study report, 2014). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 60 mg of test substance in 300 ml of BBM to get the final concentration of 200 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50, 100 and 200 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while following changes were observed at the concentration of 200 mg/l which includes, decrease in cell count, discoloration of algal cells, deformed and shrinked cell size and fine particles observed inside the cell. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 193.43 and 200 mg/l. On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

 

In a prediction done using EPI Suite ECOSAR version 1.11, the toxicity of the test chemical to green algae was predicted. On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 75.211 mg/l. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

For the test chemical, algal toxicity study was carried out for 72 hrs for assessing the effect of test chemical (authoritative database, 2020). The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Study was carried out under static conditions. Based on the effect of test chemical on growth rate of the test algae, the 72 hrs NOEC and EC50 value was determined to be 26 mg/l & 57 mg/l, respectively and on the basis of AUG, the 72 hrs NOEC and EC50 value was determined to be 24 mg/l & 41 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae.Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.