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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 (%) 
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 
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)
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 (%) 
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 
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)
Endpoint:
toxicity to microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key 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
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).
Endpoint:
toxicity to microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key 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).
Endpoint:
toxicity to microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key 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).

Description of key information

In activated sludge respiration inhibition tests, effects of sodium sulfite and ammonium thiosulfate were not observed at the limit concentration of 1000 mg test item/L. Furthermore, potassium as essential element has a very low potential for toxicity to aquatic microorganisms. Based on read-across to sulfite/disulfite/thiosulfate substances and soluble potassium substances and taking into account ubiquitousness and essentiality of potassium and sulfur, the potential of potassium sulfite for toxicity to STP microorganisms can be expected to be very low.

Key value for chemical safety assessment

Additional information

One study on the toxicity of potassium sulfite to Pseudomonas putida is available (BASF, 1988a). However, no guideline-conform activated sludge respiration inhibition test is available with potassium sulfite, thus, read-across to sulfite/disulfite and thiosulfate substances is applied. Upon contact with water, salts of sulfur oxyacids including potassium sulfite dissociate into sulfur oxyacid anions and the respective counterions. Only the properties of the sulfite anion are considered relevant determinants of the respective environmental toxicity since potassium cations are essential and have a very low potential for toxicity to freshwater organisms. Please refer to the endpoint summary for potassium (K) for further details.

Sulfite/disulfite and thiosulfate substances – Toxicity to microorganisms

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 dipotassium disulfite (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 SO32-/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 SO32-/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. Thus, the potential of sulfite/disulfite and thiosulfate substances for toxicity to STP microorganisms can be expected to be very low.