Hydroxylamine-O-sulphonic acid

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:

25.09.2017 - 28.09.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to assess the toxic effects of the test compound Hydroxylamine-O-sulphonic acid on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (IUPAC name): Hydroxylamine-O-sulphonic acid
- Molecular formula: H3NO4S
- Molecular weight: 113.093 g/mol
- Smiles notation: O=S(=O)(ON)O
- InChl: 1S/H3NO4S/c1-5-6(2,3)4/h1H2,(H,2,3,4)
- Substance type: Organic
- Physical state: Solid
- Batch No. H1238
- Physical Appearance White powder,Hygroscopic
- Storage Conditions 1. Ambient Temp (23 to 27 °C)
- Precautions Wear respiratory protection. Avoid dust formation. Avoid breathing dust, fume, vapours, mist, spray or gas. Ensure adequate ventilation. Evacuate personnel to safe areas. Use only in a chemical fume hood.

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Method: The test solution was prepared in aseptic condition. The test item Hydroxylamine-O-sulphonic acid was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/sonication for 0 minutes 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.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (Laboratory)
- Method of cultivation: Bold’s Basal Medium(BBM)


ACCLIMATION
- Culturing media and conditions (same as test or not):The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, 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
- Any deformed or abnormal cells observed:no

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

24, 48, 72 hrs

Test temperature:

22 °C±2°C.

Nominal and measured concentrations:

Six test concentration were 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l, 100mg/l, 200mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: Conical flasks of 100 ml size filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, 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: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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 data
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms


Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

152.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Nontoxic

Reported statistics and error estimates:

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.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and	24 Hours	48 Hours	72 Hours
test concentration
Control
Replicate1	18400	24000	33200
Replicate2	17600	21600	30800
Replicate3	19600	22400	34800
CAS No. 2950-43-8
6.25mg/l
Replicate1	16400	24000	31200
Replicate2	14000	21600	28000
12.5mg/l
Replicate1	13600	18400	22400
Replicate2	13600	17600	26400
25mg/l
Replicate1	13200	16400	18800
Replicate2	12400	15200	15600
50mg/l
Replicate1	14000	14400	17200
Replicate2	12000	12800	16400
100mg/l
Replicate1	12400	13600	14800
Replicate2	10800	12400	15600
200mg/l
Replicate1	11600	12800	14000
Replicate2	10400	11600	12800

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/l		200mg/l
Average	R1	0.40	R1	0.38	R1	0.36	R1	0.30	R1	0.31	R1	0.24	R1	0.16
Specific	R2	0.37	R2	0.40	R2	0.37	R2	0.31	R2	0.26	R2	0.27	R2	0.20
Growth rate (µR3		0.42
)
Mean of Avg.
Specific		0.40		0.39		0.37		0.31		0.29		0.25		0.18
growth rate
Percentage		_		1.80		7.76		23.04		27.41		36.19		53.62
Inhibition (%I)

Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and	0 Hours	72 Hours
test concentration
CONTROL
Replicate1	6.7	6.68
Replicate2	6.76	6.65
Replicate3	6.78	6.67
Average	6.75	6.67
CAS No.2950-43-8
6.25mg/l
Replicate1	5.48	6.57
Replicate2	6.09	6.58
12.5mg/l
Replicate1	6.48	6.46
Replicate2	6.47	6.49
25mg/l
Replicate1	6.20	6.26
Replicate2	6.16	6.29
50mg/l
Replicate1	5.64	5.81
Replicate2	5.2	5.75
100mg/l
Replicate1	4.30	4.73
Replicate2	4.52	4.71
200mg/l
Replicate1	3.20	3.37
Replicate2	3.22	3.33

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item Hydroxylamine-O-sulphonic acid to various nominal test concentrations, EC50 was determine to be 152.78 mg/l graphically and through probit analysis.

Executive summary:

This study was designed to assess the toxic effects of the test compound Hydroxylamine-O-sulphonic acid on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201. Chemical was analytically moniorized by spectrophotometer. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l.

All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test item Hydroxylamine-O-sulphonic acid was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/sonication for 0 minutes 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.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined.

After 72 hours of exposure to test item Hydroxylamine-O-sulphonic acid to various nominal test concentrations, EC50 was determine to be 152.78 mg/l graphically and through probit analysis. Based on the EC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Description of key information

After 72 hours of exposure to test item Hydroxylamine-O-sulphonic acid to various nominal test concentrations, EC50 was determine to be 152.78 mg/l graphically and through probit analysis.

Key value for chemical safety assessment

EC50 for freshwater algae:

152.78 mg/L

Additional information

This study was designed to assess the toxic effects of the test compound Hydroxylamine-O-sulphonic acid on the green alga Chlorella vulgaris (From experimental study report 2018). Test was conducted in compliance with the OECD guideline 201. Chemical was analytically moniorized by spectrophotometer. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l. All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test item Hydroxylamine-O-sulphonic acid was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/sonication for 0 minutes 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. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. After 72 hours of exposure to test item Hydroxylamine-O-sulphonic acid to various nominal test concentrations, EC50 was determine to be 152.78 mg/l graphically and through probit analysis. Based on the EC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.