Sodium dithionite

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented study which meets basic scientific principles.

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Principles of method if other than guideline:

growth inhibition test using a mixture of aerobic bacteria

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: other bacteria: mixed cultures

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Duration:

16 h

Dose descriptor:

IC50

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks:

based on turbidity measurements

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-06-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Principles of method if other than guideline:

German Industrial Standard DIN 38412, Part 8

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes, Pseudomonas putida, DSM 50026
- Method of cultivation: liquid
- Pretreatment/Pre- culture: Incubated at 24 °C (297 K +- 1 K), 150 rpm for 7+-1 h, Medium: AK-medium according to DIN 38412, Part 8 (draft) , Test vessel: 300 ml-Erlenmeyer flasks, 1 baffle, Liquid volume: 100 ml
- Initial biomass concentration: 1 ml pre-culture (adjusted to 10 TE/F)

Test type:

static

Water media type:

freshwater

Total exposure duration:

17 h

Test temperature:

20°C

pH:

see below

Dissolved oxygen:

see below

Nominal and measured concentrations:

Test concentrations (nominal): 15.63, 31.25, 62.5, 125, 250, 500 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Penicillium glass vessel, Liquid volume 10 ml
- No. of organisms per vessel: 1 ml pre-culture (adjusted to 10 TE/F)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control inoculated (replicates): 4
- No. of vessels non inoculated (replicates): 1 per concentration and control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ak-medium according to DIN 38412, Part 8 (draft)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- photometric determination at 436 nm and pH at test start and after 17 h

TEST CONCENTRATIONS
- Test concentrations: 15.63, 31.25, 62.5, 125, 250, 500 and 1000 mg/L.

Duration:

17 h

Dose descriptor:

other: EC90

Effect conc.:

219.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell multiplication

Duration:

17 h

Dose descriptor:

EC10

Effect conc.:

61.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell multiplication

Duration:

17 h

Dose descriptor:

EC50

Effect conc.:

106.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell multiplication

- pH at the start (0 h; w/o cells) and after 17 h (w cells):
    concentration (mg/L)   pH (0 h)     pH (17 h)
         15.63               7.0           6.4  
         31.25               6.9           6.7
         62.5                6.8           6.9
        125.0                6.6           6.8
        250.0                6.4           6.4
        500.0                6.1           6.0
       1000.0                5.7           5.4
       control               7.0           5.1

- Oxygen (mg/L) at the start (0 h; w cells) and after 17 h
(w cells): 
    concentration (mg/L)   O2 (0 h)    O2 (17 h)
         15.63               8.2         0.7
       1000.0                8.6         8.8

Conclusions:

A growth inhibition test with the micro-organism Pseudomonas putida was conducted, following standard guidelines (non-GLP). Reported 17h-EC50 and EC10 were 106.5 and 61.6 mg test substance/L, respectively. Data can be used as supporting information for the micro-organisms effects assessment of hydrosulfite (dithionite) and other dithionite compounds (read-across principle).

Reference 3

Endpoint:

toxicity to microorganisms

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1988-07-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP Guideline study without analytical verification of test concentrations

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared dissolving 1250 mg of the test substance in 1 l of deionized water. From the stock solution 9 concentrations were prepared through 1:2 dilution steps.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida regularly obtained from DSM (German collection of microorganisms, Goettingen, Germany)
- strain: DSM 50026
- Pre-culture:
- Test vessel: Erlenmeyer flasks, volume 300 mL, fill volume 100 mL
- inoculated with bacterial suspension at a turbidity of 10 TE/F at 21 ± 1 °C
- incubation time: 7 hours, shaking
- nutrient medium: AK-Medium, DIN 38412 Part 8

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 h

Test temperature:

20°C

pH:

test start: 5.3 - 7.0 mg/L
test end: 4.6 - 7.0 mg/L (inoculated)
test end: 4.7 - 7.2 mg/L (without inoculum)

Dissolved oxygen:

test start: 5.0 - 9.2 mg/L
test end: 0.3 - 8.8 mg/L (inoculated)
test end: 8.8-8.9 mg/L (without inoculum)

Nominal and measured concentrations:

0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Penicillium vessel with flattened bottom, plugged with silicone sponge caps
- Material, fill volume: glass, 10 mL
- Aeration: no
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient medium: 0.5 g/L Sodium nitrate, 0.12 g/L dipotassium hydrogen phosphate, 0.06 g/L Potassium dihydrogen phosphate, 0.2 g/L magnesium sulphate heptahydrate, 2.0 g/L D(+)-Glucose, 0.0005 g/L ferric citrate

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Photometric determination of light extinction at 436 nm after 17 hours of exposure
- pH and oxygen was measured at test start end test end in inoculated samples and blank medium, respectively.

Duration:

17 h

Dose descriptor:

other: EC90

Effect conc.:

115.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

EC10

Effect conc.:

30.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

EC50

Effect conc.:

56.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Optical density compared to the control (=100%)

Conc [mg/L]	Optical density	% of control
0		100
7.81	0.394	100.51
15.63	0.396	100.89
31.25	0.352	89.67
62.5	0.156	39.83
125	0.017	4.4
250	0.002	0.38
500	0.003	0.64
1000	0.002	0.51

Conclusions:

A growth inhibition test with the micro-organism Pseudomonas putida was conducted, following standard guidelines (non-GLP). Reported 17h-EC50 and EC10 were 56.1 and 30.8 mg test substance/L, respectively. Data can be used as supporting information for the micro-organisms effects assessment of disodium disulfite and other sulfite compounds (read-across principle).

Reference 4

Endpoint:

toxicity to microorganisms

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1988-07-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP Guideline study without analytical verification of test concentrations

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared dissolving 1250 mg of the test substance in 1 l of deionized water. From the stock solution 9 concentrations were prepared through 1:2 dilution steps.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida regularly obtained from DSM (German collection of microorganisms, Goettingen, Germany)
- strain: DSM 50026
- Pre-culture:
- Test vessel: Erlenmeyer flasks, volume 300 mL, fill volume 100 mL
- inoculated with bacterial suspension at a turbidity of 10 TE/F at 21 ± 1 °C
- incubation time: 7 hours, shaking
- nutrient medium: AK-Medium, DIN 38412 Part 8

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 h

Test temperature:

20°C

pH:

test start: 7.0 - 7.9 mg/L
test end: 5.0 - 7.4 mg/L (inoculated)
test end: 7.0 - 7.6 mg/L (without inoculum)

Dissolved oxygen:

test start: 7.1 - 8.5 mg/L
test end: 0.3 - 5.6 mg/L (inoculated)
test end: 9.0-9.1 mg/L (without inoculum)

Nominal and measured concentrations:

0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Penicillium vessel with flattened bottom, plugged with silicone sponge caps
- Material, fill volume: glass, 10 mL
- Aeration: no
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient medium: 0.5 g/L Sodium nitrate, 0.12 g/L dipotassium hydrogen phosphate, 0.06 g/L Potassium dihydrogen phosphate, 0.2 g/L magnesium sulphate heptahydrate, 2.0 g/L D(+)-Glucose, 0.0005 g/L ferric citrate

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Photometric determination of light extinction at 436 nm after 17 hours of exposure
- pH and oxygen was measured at test start end test end in inoculated samples and blank medium, respectively.

Duration:

17 h

Dose descriptor:

EC10

Effect conc.:

153 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

EC50

Effect conc.:

410 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Optical density compared to the control (=100%)

Conc [mg/L]	Optical density	% of control
0		100
7.81	0.443	98.06
15.63	0.482	106.7
31.25	0.496	109.74
62.5	0.51	112.84
125	0.438	96.85
250	0.301	66.52
500	0.184	40.79
1000	0.122	27.06

Conclusions:

A growth inhibition test with the micro-organism Pseudomonas putida was conducted, following standard guidelines (non-GLP). Reported 17h-EC50 and EC10 were 410 and 153 mg test substance/L, respectively. Data can be used as supporting information for the micro-organisms effects assessment of potassium sulfite and other sulfite compounds (read-across principle).

Reference 5

Endpoint:

toxicity to microorganisms

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

1988-07-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP Guideline study without analytical verification of test concentrations

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared dissolving 1250 mg of the test substance in 1 l of deionized water. From the stock solution 9 concentrations were prepared through 1:2 dilution steps.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida regularly obtained from DSM (German collection of microorganisms, Goettingen, Germany)
- strain: DSM 50026
- Pre-culture:
- Test vessel: Erlenmeyer flasks, volume 300 mL, fill volume 100 mL
- inoculated with bacterial suspension at a turbidity of 10 TE/F at 21 ± 1 °C
- incubation time: 7 hours, shaking
- nutrient medium: AK-Medium, DIN 38412 Part 8

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 h

Test temperature:

20°C

pH:

test start: 4.7 - 7.0 mg/L
test end: 4.3 - 7.1 mg/L (inoculated)
test end: 4.6 - 7.2 mg/L (without inoculum)

Dissolved oxygen:

test start: 5.7 - 9.5 mg/L
test end: 0.5 - 8.8 mg/L (inoculated)
test end: 8.9-9.1 mg/L (without inoculum)

Nominal and measured concentrations:

0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Penicillium vessel with flattened bottom, plugged with silicone sponge caps
- Material, fill volume: glass, 10 mL
- Aeration: no
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient medium: 0.5 g/L Sodium nitrate, 0.12 g/L dipotassium hydrogen phosphate, 0.06 g/L Potassium dihydrogen phosphate, 0.2 g/L magnesium sulphate heptahydrate, 2.0 g/L D(+)-Glucose, 0.0005 g/L ferric citrate

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Photometric determination of light extinction at 436 nm after 17 hours of exposure
- pH and oxygen was measured at test start end test end in inoculated samples and blank medium, respectively.

Duration:

17 h

Dose descriptor:

other: EC90

Effect conc.:

124 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

EC10

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Optical density compared to the control (=100%)

Conc [mg/L]	Optical density	% of control
0		100
7.81	0.409	99.51
15.63	0.404	98.42
31.25	0.374	91.11
62.5	0.212	51.55
125	0.038	9.19
250	0.002	0.49
500	0.002	0.43
1000	0.001	0.24

Conclusions:

A growth inhibition test with the micro-organism Pseudomonas putida was conducted, following standard guidelines (non-GLP). Reported 17h-EC50 and EC10 were 64.8 and 32.1 mg test substance/L, respectively. Data can be used as supporting information for the micro-organisms effects assessment of potassium disulfite and other (di-)sulfite compounds (read-across principle).

Reference 6

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 2009-study, conducted under GLP

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A microbial inoculum was used in htis test system. Activated sludge from a sewage treatment plant was used as the microbial inoculum for the test. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.
- Date of sampling/date of arrival at ECT: August 16, 2010
- Washing medium for activated sludge: reconstituted water (OECD 1992)
- Frequency of washing: three times - between washings the activated sludge was centrifuged for 2 min at 1000 rpm
- Storage before use: 4 days
- Storage temperature: 20 +/- 2 degrees Celcius
- pH of the activated sludge: 6.9
- mixed liquor suspended solids level in the microbial inoculum: 4.36 g/L
Reconstituted water according to OECD Guideline No. 203 (Fish, acute toxicity test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations: CaCl2.2H2O: 294 mg/L ; MgSO4.7H2O: 123.25 mg/L ; NaHCO3: 64.75 mg/L ; KCl: 5.75 mg/L ; pH: 7.8 ; Hardness: 253.6 mg/L as CaCO3 ; O2-conc.: 9 mg/L. Medium was prepared within one month before use, and was aerated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.0 to 20.2 degrees Celcius (n=29)

pH:

pH of activated sludge: 6.9

Nominal and measured concentrations:

Definitive test: 5 concentration levels + control
nominal levels: 0, 10, 32, 100, 316 and 1000 mg test item/L

Details on test conditions:

Preparation of test solutions: Stock solution was prepared by dissolving 1.0003g of the test item in 500 mL of deionised water. Stock solution was stirred on a magnetic stirring device for approx. 5 min and during removal of corresponding amounts of stock solution for preparation of each single test vessel.
- method of administration of the inoculum was based on the requirements of the test guideline
* amount of synthetic sewage feed in test solution: 16 mL
* amount of test solution/concentration: 300 ml
* amount of microbial inoculum added: 200 mL
* total test volume: 500 mL
* mixed liquor suspended solids level in the final test mixtures: 1.74 g/L
- two replicates were used for the control
- vessels were prepared at intervals of 15 minutes, starting with the first control, followed by test item concentration levels and the second control at the end
- before use, vessels were sterilized in a drying oven for three hours at 150 degrees Celcius.
- type of vessel: 1000 mL glass beakers without cover
- one replicate for each concentration level was tested
- aeration of test vessels: 40 to 50 liter of air per hour (ambient air, oil-free air-compressor)
- at the end of the contact time (= test period) the content of the first vessel was poured into the measuring apparatus and the respiration rate was determined. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.

Synthetic sewage feed: the following amounts of substances in 1L of deionized water (in g/L) - prepared within 1 week before use:
* peptone: 16
* meat extract: 11
* urea: 3
* NaCl: 0.7
* CaCl2.2H2O: 0.4
* MgSO4.7H2O: 0.2
* K2HPO4: 2.8

Oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. Oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The control respiration rates did not differ more than 15% compared to each other, i.e., only 2.9%

Results with reference substance (positive control):

The 3h-EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L, i.e., 10.0 mg/L

Reported statistics and error estimates:

Respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L between 6.5 and 2.5 mg O2/L.
The portion of the respiration curve over which the respiration rate was measured was linear.
Respiration rate was expressed as percentage of the mean of the two control respiration rates, thus allowing to determine ECx values
Weibull Analysis was used to determine the ECx values. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations

Concentration (mg/L	Oxygen consumption (mg/L per hour)	Inhibition (%)
0	68.28	2.03
10	68.40	1.85
32	71.10	-2.03
100	70.07	-0.55
316	72.85	-4.54
1000	71.27	-2.26
0	71.10	-2.03

Validity criteria fulfilled:

yes

Conclusions:

An OECD No. 209 was performed, using ammonium thiosulfate as test substance. The test was conducted under GLP. Inhibition of respiration rate after a 3h-exposure period was evaluated. No significant adverse effects were noted up to the highest test concentration of 1000 mg/L, resulting in a NOEC value equal or higher than 1000 mg test substance/L .
Data can be used for the effects assessment of ammonium thiosulfate and other thiosulfate compounds (read-across principle)

Reference 7

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 2009-study, conducted under GLP

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A microbial inoculum was used in htis test system. Activated sludge from a sewage treatment plant was used as the microbial inoculum for the test. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.
- Date of sampling/date of arrival at ECT: August 16, 2010
- Washing medium for activated sludge: reconstituted water (OECD 1992)
- Frequency of washing: three times - between washings the activated sludge was centrifuged for 2 min at 1000 rpm
- Storage before use: 4 days
- Storage temperature: 20 +/- 2 degrees Celcius
- pH of the activated sludge: 6.9
- mixed liquor suspended solids level in the microbial inoculum: 4.36 g/L
Reconstituted water according to OECD Guideline No. 203 (Fish, acute toxicity test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations: CaCl2.2H2O: 294 mg/L ; MgSO4.7H2O: 123.25 mg/L ; NaHCO3: 64.75 mg/L ; KCl: 5.75 mg/L ; pH: 7.8 ; Hardness: 253.6 mg/L as CaCO3 ; O2-conc.: 9 mg/L. Medium was prepared within one month before use, and was aerated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.0 to 20.2 degrees Celcius (n=29)

pH:

pH of activated sludge: 6.9

Nominal and measured concentrations:

Definitive test: 5 concentration levels + control
nominal levels: 0, 10, 32, 100, 316 and 1000 mg test item/L

Details on test conditions:

Preparation of test solutions: Stock solution was prepared by dissolving 1.0003g of the test item in 500 mL of deionised water. Stock solution was stirred on a magnetic stirring device for approx. 5 min and during removal of corresponding amounts of stock solution for preparation of each single test vessel.
- method of administration of the inoculum was based on the requirements of the test guideline
* amount of synthetic sewage feed in test solution: 16 mL
* amount of test solution/concentration: 300 ml
* amount of microbial inoculum added: 200 mL
* total test volume: 500 mL
* mixed liquor suspended solids level in the final test mixtures: 1.74 g/L
- two replicates were used for the control
- vessels were prepared at intervals of 15 minutes, starting with the first control, followed by test item concentration levels and the second control at the end
- before use, vessels were sterilized in a drying oven for three hours at 150 degrees Celcius.
- type of vessel: 1000 mL glass beakers without cover
- one replicate for each concentration level was tested
- aeration of test vessels: 40 to 50 liter of air per hour (ambient air, oil-free air-compressor)
- at the end of the contact time (= test period) the content of the first vessel was poured into the measuring apparatus and the respiration rate was determined. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.

Synthetic sewage feed: the following amounts of substances in 1L of deionized water (in g/L) - prepared within 1 week before use:
* peptone: 16
* meat extract: 11
* urea: 3
* NaCl: 0.7
* CaCl2.2H2O: 0.4
* MgSO4.7H2O: 0.2
* K2HPO4: 2.8

Oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. Oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

187.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 171.6-203.7

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

89.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 75.2-103.1

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

120.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 105.2-134.3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

260.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 239.9-285.7

Details on results:

The control respiration rates did not differ more than 15% compared to each other, i.e., only 3.2%.
A clear concentration-response relationship was observed The respiration rates at the two highest treatment levels were based between 8.31 O2/L and 9.34 O2/L, whcih is outside the range of 6.5 mg O2/L to 2.5 mg O2/L that is frecommended in the guideline. This was done due to low respiration rates encountered at these treatment levels. shifting the range allowed calculation of respiratrion rates.

Results with reference substance (positive control):

The 3h-EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L, i.e., 10.0 mg/L

Reported statistics and error estimates:

Respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L between 6.5 and 2.5 mg O2/L.
The portion of the respiration curve over which the respiration rate was measured was linear.
Respiration rate was expressed as percentage of the mean of the two control respiration rates, thus allowing to determine ECx values
Weibull Analysis was used to determine the ECx values. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations

Concentration (mg/L)	Oxygen consumption (mg/L per hour)	Inhibition (%)
0	82.48	2.28
10	81.76	3.12
32	108.75	-28.85
100	74.28	11.99
316	5.66	93.29
1000	0.79	99.06
0	86.32	-2.28

Validity criteria fulfilled:

yes

Conclusions:

An OECD No. 209 was performed, using sodium dithionite as test substance. The test was conducted under GLP. Inhibition of respiration rate after a 3h-exposure period was evaluated. And EC50 and EC20 of 187.6 mg/L and 120.5 mg/L, respectively, were determined. Data can be used for the effects assessment of sodium dithionite and other dithionite compounds (read-across principle)

Reference 8

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 2009-study, conducted under GLP

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A microbial inoculum was used in htis test system. Activated sludge from a sewage treatment plant was used as the microbial inoculum for the test. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.
- Date of sampling/date of arrival at ECT: August 16, 2010
- Washing medium for activated sludge: reconstituted water (OECD 1992)
- Frequency of washing: three times - between washings the activated sludge was centrifuged for 2 min at 1000 rpm
- Storage before use: 4 days
- Storage temperature: 20 +/- 2 degrees Celcius
- pH of the activated sludge: 6.9
- mixed liquor suspended solids level in the microbial inoculum: 4.36 g/L
Reconstituted water according to OECD Guideline No. 203 (Fish, acute toxicity test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations: CaCl2.2H2O: 294 mg/L ; MgSO4.7H2O: 123.25 mg/L ; NaHCO3: 64.75 mg/L ; KCl: 5.75 mg/L ; pH: 7.8 ; Hardness: 253.6 mg/L as CaCO3 ; O2-conc.: 9 mg/L. Medium was prepared within one month before use, and was aerated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.0 to 20.2 degrees Celcius (n=29)

pH:

pH of activated sludge: 6.9

Nominal and measured concentrations:

Definitive test: 5 concentration levels + control
nominal levels: 0, 10, 32, 100, 316 and 1000 mg test item/L

Details on test conditions:

Preparation of test solutions: Stock solution was prepared by dissolving 1.0003g of the test item in 500 mL of deionised water. Stock solution was stirred on a magnetic stirring device for approx. 5 min and during removal of corresponding amounts of stock solution for preparation of each single test vessel.
- method of administration of the inoculum was based on the requirements of the test guideline
* amount of synthetic sewage feed in test solution: 16 mL
* amount of test solution/concentration: 300 ml
* amount of microbial inoculum added: 200 mL
* total test volume: 500 mL
* mixed liquor suspended solids level in the final test mixtures: 1.74 g/L
- two replicates were used for the control
- vessels were prepared at intervals of 15 minutes, starting with the first control, followed by test item concentration levels and the second control at the end
- before use, vessels were sterilized in a drying oven for three hours at 150 degrees Celcius.
- type of vessel: 1000 mL glass beakers without cover
- one replicate for each concentration level was tested
- aeration of test vessels: 40 to 50 liter of air per hour (ambient air, oil-free air-compressor)
- at the end of the contact time (= test period) the content of the first vessel was poured into the measuring apparatus and the respiration rate was determined. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.

Synthetic sewage feed: the following amounts of substances in 1L of deionized water (in g/L) - prepared within 1 week before use:
* peptone: 16
* meat extract: 11
* urea: 3
* NaCl: 0.7
* CaCl2.2H2O: 0.4
* MgSO4.7H2O: 0.2
* K2HPO4: 2.8

Oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. Oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The control respiration rates did not differ more than 15% compared to each other, i.e., only 0.6%

Results with reference substance (positive control):

The 3h-EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L, i.e., 10.0 mg/L

Reported statistics and error estimates:

Respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L between 6.5 and 2.5 mg O2/L.
The portion of the respiration curve over which the respiration rate was measured was linear.
Respiration rate was expressed as percentage of the mean of the two control respiration rates, thus allowing to determine ECx values
Weibull Analysis was used to determine the ECx values. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations

Concentration (mg/L)	Oxygen consumption (mg/L per hour)	Inhibition (%)
0	82.88	0.46
10	84.21	-1.14
32	111.5	-33.91
100	104.95	-26.04
316	90.46	-8.64
1000	86.00	-3.28
0	83.65	-0.46

Validity criteria fulfilled:

yes

Conclusions:

An OECD No. 209 was performed, using sodium sulfite as test substance. The test was conducted under GLP. Inhibition of respiration rate after a 3h-exposure period was evaluated. No significant adverse effects were noted up to the highest test concentration of 1000 mg/L, resulting in a NOEC value equal or higher than 1000 mg test substance/L .
Data can be used for the effects assessment of sodium sulfite and other (di-)sulfite compounds (read-across principle)

Reference 9

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well documented publication which meets basic scientific principles

Analytical monitoring:

no

Details on test conditions:

Type: other: species: bacteriophage phi X174

Duration:

2 h

Dose descriptor:

EC0

Effect conc.:

99.2 mg/L

RS-Freetext:
- Bacteriophage phi X174 was inactivated by mitomycin C reduced with  sodium hydrosulfite in the presence of cupric ions (Cu2++). 

- 99 % of the phage particles lost their plaque-forming abilities when  incubated with 1.5*10-4 M mitomycin C, 5.7*10-4 M sodium hydrosulfite and  1.0*10-4 M CuCl2 for 120 min at 37 °C in 0.05 M Tris-HCl buffer (pH 8.1).

- Sodium borohydride and thiol-reducing agents such as L-cysteine,  2-mercaptoethanol or dithiothreitol could not serve as a substitute for  sodium hydrosulfite.

- Strand-scission was observed when phi X174 single-stranded DNA was  directly reacted with mitomycin C reduced with sodium hydrosulfite in the  presence of CuCl2.

Description of key information

In an activated sludge respiration inhibition tests according to OECD 209 with sodium dithionite, 3-h EC10 and EC50 values of 89.8 mg/L and 187.6 mg/L, respectively, were derived for inhibition of total respiration/respiration rate.

Key value for chemical safety assessment

EC50 for microorganisms:

187.6 mg/L

EC10 or NOEC for microorganisms:

89.8 mg/L

Additional information

One study on the toxicity of sodium dithionite to aquatic microorganisms is available (activated sludge, ECT, 2010). In addition, read-across to thiosulfate and sulfite/disulfite substances is applied. Upon contact with water, salts of sulfur oxyacids including sodium dithionite dissociate into sulfur oxyacid anions and the respective counterions. Only the properties of the dithionite anion are considered a relevant determinant of environmental toxicity since sodium cations are essential and have a very low potential for toxicity to aquatic microorganisms. Please refer to the respective endpoint summary for sodium (Na) for further details. Dithionite anions are unstable under environmentally relevant conditions and will rapidly disproportionate to (bi-)sulfites and thiosulfates (S₂O₃^2-) in aqueous media.Sulfites and thiosulfates are unstable in the environment, subject to transformation processes including oxidation to sulfate or reduction to sulfide, and will ultimately become part of the natural sulfur cycle.

 

Dithionite, sulfite/disulfite and thiosulfate substances – Toxicity to microorganisms

In a reliable OECD No. 209 respiration inhibition test (under GLP) with sodium dithionite (Na2SO4), 3-h EC10 and EC50 values of 89.8 mg/L and 187.6 mg/L, respectively, were derived for the inhibition of the respiration rate of activated sludge. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.

 

In addition, a reliable OECD No. 209 respiration inhibition test (under GLP) was performed with sodium sulfite (Na2SO3). The activated sludge originates from a sewage treatment plant in Frankfurt/Main Niederrad treating predominantly domestic sewage. Effects on respiration were not observed at the highest test concentration of 1000 mg test substance/L, and the 3h-NOEC expressed as sulfite (SO32-) amounts to ≥ 634.4 mg/L.

 

Furthermore, an OECD No. 209 respiration inhibition test (under GLP) was performed with ammonium thiosulfate. Significant adverse effects were not observed up to the highest test concentration of 1000 mg/L after 3 h, resulting in a NOEC value ≥ 1000 mg/L (corresponding to ≥ 540.2 mg SO32-/L).

 

The absence of any toxicity at this level is confirmed by a 16h-EC50 of 2000 mg NaSO3/L (corresponding to 1268 mg SO32-) determined in a growth inhibition test on a mixed culture of bacteria by Alsop et al. (1980).

 

Additionally, the growth inhibition of the bacterium Pseudomonas putida by three different sulfite substances (i.e., potassium sulfite (K2SO3), disodium disulfite (Na2S2O5) and potassium metabisulfite (K2S2O5)) was tested and the following effect levels were determined:

Substance	MM	% S	17 h-EC50(mg test item/L)	17 h-EC50(mg S/L)	17 h-EC50(mg SO3^2-/L)	Reference
K2SO3	158.26	20.3	410	83.1	207.2	BASF, 1988a
K2S2O5	222.32	28.8	65	18.8	46.8	BASF, 1988b
Na2S2O5	190.1	33.7	56.1	18.9	47.2	BASF, 1988c
			17 h-EC10(mg test item/L)	17 h-EC10(mg S/L)	17 h-EC10(mg SO3^2-/L)
K2SO3	158.26	20.3	153	31	77.3	BASF, 1988a
K2S2O5	222.32	28.8	32	9.2	23.0	BASF, 1988b
Na2S2O5	190.1	33.7	30.8	10.4	25.9	BASF, 1988c

 

ECHA Guidance on IR & CSA, Chapter 7b: Endpoint Specific Guidance (Version 4.0, 2017) states that “Results of the cell multiplication inhibition test with P. putida (Bringmann and Kühn 1980) should be used for calculation of the PNECmicro-organisms only in cases where no other test results are available.” Thus, results are considered as supporting only and the hazard assessment is based on results from activated sludge respiration inhibition tests.

 

In activated sludge respiration inhibition tests, effects of sodium sulfite and ammonium thiosulfate were not observed at the highest test concentration of 1000 mg test item/L, the OECD test limit, respectively. In an activated sludge respiration inhibition tests with sodium dithionite, 3-h EC10 and EC50 values of 89.8 mg/L and 187.6 mg/L, respectively, were derived for inhibition of total respiration/respiration rate and are applied in the hazard assessment of sodium dithionite.