Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental results of read across chemicals

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE report is prepared based on short term toxicity to aquatic algae study.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine
- Molecular formula: C11H23N7O3S
- Molecular weight: 333.41 g/mol
- Smiles notation: C=O_CC1CCC(S(N)(=O)=O)CC1_Nc1nc(N)nc(N)n1
- Substance type: Organic
- Physical state: Solid

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

1. PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10E4cells/ml. Care was taken to have a homogeneous solution for the experiment.

2. The stock solution (150 mg/L) was prepared by dissolving white powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 min.Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

3. The stock solution 150 mg/l was prepared by dissolving light brown powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 min. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

other: 1. Chlorella vulgaris, 2, 3. Desmodesmus subspicatus

Details on test organisms:

1.TEST ORGANISM
- Strain: No data
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur
- Age of inoculum (at test initiation): 1x10E4 cell/ml
- Method of cultivation: no data


2, 3. TEST ORGANISM:
- Common name:
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5x10(3) cells /ml

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Post exposure observation period:

24, 48 and 72 hrs.

Test temperature:

1. 22 ° C ± 2°C.
2. 23±2°C

pH:

Test pH varies with the different concentrations: 7.9 - 5
Control 1: 8.3 (changed to 7.9 during test)
Control 2: 8 (changed to 7.8 during test)

Nominal and measured concentrations:

1. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l, 100mg/l, 200mg/l
2 and 3. 0, 0, 30, 45, 67, 100, 150 mg/l.

Details on test conditions:

1. TEST SYSTEM
- Test vessel: All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized.
- Material, size, headspace, fill volume: 60ml
- Initial cells density: 13.625 x10E4 cells/mL
- No. of vessels per concentration (replicates): Two replicates
- No. of vessels per control (replicates): Three replicates


GROWTH MEDIUM
- Standard medium used: Yes
- The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrien ts, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: White Fluorescent Light (1500Lux)
- RPM speed: 120 Revolutions per minute

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell counting: Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer.
Microscopic observations : The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.
Spectrophotometer: The absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.
- Chlorophyll measurement: No
- Other: EC50 value was determined.

TEST CONCENTRATIONS
- Test concentrations: Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l


2 and 3.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

Reference substance (positive control):

yes

Remarks:

In 2 and 3 only Potassium dichromate (K2Cr2O7) were used as a positive control

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: For 1 study

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

110.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 108.8 - 112.4 for study 2

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

110 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: For 3 study 95% CI was 100.6 - 120.2 mg/l

Details on results:

1. Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as
compared to 0 hours. The average pH drift observed in the control vessels was 0.44 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. All the six concentrations were in geometric series spaced by a factor of 2.
The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. EC50 was found to be >200 mg/l graphically through probit
analysis.

Results with reference substance (positive control):

2 and 3. Results with reference substance valid
- EC50: 0.75 mg/L

Reported statistics and error estimates:

1. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.
2 and 3. ErC50 was calculated using non-linear regression by the software Prism 4.0

1:

Table 1: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		6.25 mg/l			12.5 mg/l			25 mg/l			50 mg/l			100 mg/l			200 mg/l
Average Specific	R1	0.608		R1	0.423		R1	0.419		R1	0.400		R1	0.375		R1	0.353		R1	0.319
Growth rate (μ )	R2	0.586		R2	0.441		R2	0.408		R2	0.392		R2	0.357		R2	0.329		R2	0.297
	R3	0.612
Mean of Avg. Specific growth rate	0.602		0.432			0.414			0.396			0.366			0.341			0.308
Percentage Inhibition (%I)	_		28.202			31.301			34.254			39.208			43.440			48.868

Table 2: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and test concentration	0 Hours	72 Hours
CONTROL
Replicate1	6.99	7.37
Replicate2	6.98	7.43
Replicate3	6.99	7.50
Average	6.99	7.43
Test chemical
6.25mg/l
Replicate1	7.09	7.06
Replicate2	7.10	7.08
12.5mg/l
Replicate1	7.08	7.20
Replicate2	7.12	7.22
25mg/l
Replicate1	7.14	7.33
Replicate2	7.16	7.30
50mg/l
Replicate1	7.11	7.35
Replicate2	7.13	7.31
100mg/l
Replicate1	6.96	7.29
Replicate2	6.92	7.30
200mg/l
Replicate1	6.64	6.98
Replicate2	6.62	6.95

For 3.

Fig: pH of test:

Sample concentration in mg/l	30	45	67	100	150
Start of the test	7.8	7.0	6.5	5.7	5.2
End of the test	7.7	6.5	5.7	5.5	5.0

Validity criteria fulfilled:

yes

Conclusions:

1. After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis.
2. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 110.6 mg/L on the basis of effects on growth rate in a 72 hour study.
3. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 110 mg/L on the basis of effects on growth rate in a 72 hour study.

Executive summary:

Short term toxicity of Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine (97808 -67 -8) on the growth and other biological activity of aquatic algae is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Short term toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the algae. The effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be >200 mg/L. Thus, based on this EC50 value and according to CLP Criteria for aquatic classification of the substance, it is concluded that test chemical does not exhibits toxicity to aquatic algae (Chlorella vulgaris).

Similar study was performed for another structurally and functionally similar read across chemicals. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 0, 30, 45, 67, 100, 150 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 110.6 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Study was performed for another structurally and functionally similar read across chemicals. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution 150 mg/l was prepared by dissolving light brown powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 min. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Sample tested at the concentrations 0, 0, 30, 45, 67, 100 and 150 mg/L. Test performed by the static system for 72hrs of exposure period. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 110 mg/L on the basis of effects on growth rate in a 72 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP classification criteria.

Thus based on the overall studies for the structurally and functionally similar read across chemical for Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine (97808 -67 -8), it can be concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.  

Description of key information

1. After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis.

2. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 110.6 mg/L on the basis of effects on growth rate in a 72 hour study.

3. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 110 mg/L on the basis of effects on growth rate in a 72 hour study.

Thus based on the overall studies for Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine (97808 -67 -8), it can be concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.  

Key value for chemical safety assessment

EC50 for freshwater algae:

200 mg/L

Additional information

Short term toxicity of Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine (97808 -67 -8) on the growth and other biological activity of aquatic algae is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Short term toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the algae. The effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be >200 mg/L. Thus, based on this EC50 value and according to CLP Criteria for aquatic classification of the substance, it is concluded that test chemical does not exhibits toxicity to aquatic algae (Chlorella vulgaris).

Similar study was performed for another structurally and functionally similar read across chemicals. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 0, 30, 45, 67, 100, 150 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 110.6 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Study was performed for another structurally and functionally similar read across chemicals. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution 150 mg/l was prepared by dissolving light brown powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 min. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Sample tested at the concentrations 0, 0, 30, 45, 67, 100 and 150 mg/L. Test performed by the static system for 72hrs of exposure period. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 110 mg/L on the basis of effects on growth rate in a 72 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP classification criteria.

Thus based on the overall studies for the structurally and functionally similar read across chemical for Benzenesulfonamide, 4-methyl-, reaction products with formaldehyde and melamine (97808 -67 -8), it can be concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.