Disodium disulphite

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according German industrial standard guideline, no analytical dose verification.

Justification for type of information:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was liquidized by heating and a stock solution prepared. Preparation of the concentration range by serial dilution of stock solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus (former name: Scenedesmus subspicatus)
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Supplier: Sammlung für Algenkulturen der Universität Göttingen
- Age of inoculum (at test initiation): The test algae are taken from a 3 days-old continously growing preculture
- Method of cultivation: The test strain is kept in liquid culture in the laboratory.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20°C

pH:

test start: 4.38 - 8.2
test end: 2.8-9.8 (with inoculum)

Nominal and measured concentrations:

0, 7.81, 15.68, 31.25, 62.5, 125, 250, 500 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 20 ml glass tubes with flat bottom, plugged with siliconesponge caps, 10 ml test volume

- Initial cells density: 10000 cells/ml
- Control end cells density: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: not during test
- Photoperiod: continous

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: in vivo chlorophyll-a-fluorescence at 685 nm wavelength (as measure for cell number) after 0, 24, 48, 72 and 96 hours
- pH values: after 0 and 96 hours

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

57.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

33.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Test was conducted for 96h, and the test report provided and EC10, EC50 and EC90 for 24, 48, 72 and 96h of exposure (endpoint: biomass). ECx-values based on the more relevant endpoint "growth rate" were determined for the 72h exposure period.

Effect concentrations (mg/L) based on Chl a-fluorescence yield:

	EC20	EC50	EC90
24h	46.4	62	113.7
48h	47.3	53.7	62.3
72h	39.2	48.1	60
96h	20	39.8	58

Dissolved oxygen concentration of stock solution, directly after preparation, was 6.4 mg/L.

Table: pH in the test vessels at test start and test end

Test item concentration [mg/L]	pH (test start)	pH (96 h, inoculated)	pH (96 h, not inoculated)
0	8.2	9.8	7.9
7.8	7.5	9.8	8.0
15.6	7.27	9.5	7.9
31.25	6.97	9.1	7.7
62.5	6.6	4.9	4.8
125	6.0	3.3	3.3
250	5.4	2.9	2.9
500	4.38	2.8	2.8

Validity criteria fulfilled:

yes

Conclusions:

A 96 h algal growth inhibition toxicity test was conducted with the green alga S.subspicatus, using sodium disulfite as test compound, and 72h-EC10 and EC50 values of 33.2 and 43.9 mg test item/L were derived, respectively, corresponding to 28 and 36.8 mg SO32-/L. However, the pH in inoculated test vessels was significantly lower in test concentrations of 62.5 mg test item/L and higher, compared to the control and the lower test concentrations. Criteria for algae testing according to OECD 201 as well as OECD Series 23 ("Guidance document on aquatic toxicity testing of difficult substances and mixtures") regarding pH of the test solutions are as follows: pH in test vessels should not differ by more than 1.5 from the control medium, otherwise test cultures need to be adapted to the different conditions prior to testing, preferably all test vessels should be adjusted to ± 0.2 at test start. Furthermore, pH should not change by more than 1.5 during the exposure duration. These criteria were not fulfilled in the current study, and observed growth inhibiting effects at the level of the EC10 and the EC50 might be due to effects of pH.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: Test conducted according to OECD 201 Guideline, GLP-compliant test.

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 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata (SAG 61.81), a green alga, supplied by Georg-August-Universität Göttingen, D-37073 Göttingen, Germany.
Pre-culture conditions:
* Date of receipt: July 21, 2010
* pH-value of algal medium: 6.0
* Test medium: Algae medium m4 (Kuhl and Lorenzen, 1964)
* Amount of liquid stock culture per pre-culture vessel: 100 mL
* Pre-culture vessels: 300 mL Erlenmeyer flasks
* Number of replicates: 2
* Light: light/dark (24/0 h); fluorescent tubes of universal white type
* Light intensity: 60-120 uE/m2*s
* Shaker: 100 +/- 5 oscillations/min
* Temperature in test room: 21-24 degrees Celcius, controlled at +/- 2 degrees Celcius

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature: mean: 21.8 degrees Celcius (n=148)
min: 21.3 degrees Celcius
max: 22.5 degrees Celcius
required: 21-24 degrees Celcius controlled at +/- 2 degrees Celcius

pH:

pH at start of test: 6.0
pH at end of test: 5.8 - 6.0

Nominal and measured concentrations:

Nominal test concentrations in the final test: 0 , 1.0, 3.2, 10, 32 and 100 mg test substance/L

Details on test conditions:

Test conducted in algae medium M4 (Kuhl and Lorenzen, 1964)

After temperature adaptation of the test solutions the algal cells were transferred from the pre-culture into the test solutions. The concentration of the algal cells in the test solution at the beginning of the exposure was approximately 0.5 * 10(4) cells/mL. The method of administration is based on the requirements of the test guideline.

Test solution preparation: 200.1 mg of the test item was mixed into 2000 mL algal medium in a 2000 mL measuring flask in a nominal concentration of 100 mg test item/L. The solution was stirred for 3 minutes at room temperature.

- Age of the pre-culture: 3 d
- No. of cells/mL in the pre-culture before inoculating the test solution: 147*10(4)
- No. of cells/mL test solution at the beginning of the test: approx. 0.5 * 10(4)
- No. of replicates per test item concentration: 3
- No. of replicates in the control: 6
- Light cycle: 24/0 light/dark
- Types of light: fluorescent tubes of universal white type (Osram Lumilux 58W/865)
- Light intensity: mean value of sic measurements: 75 uE/m2*s (required: 60-120 uE/m2*s), equivalent to 4440-8880 lx)
- Shaker: 100 +/- 5 oscillations/min; the test vessels were placed randomly on the shaker
- Exposure period: 72h
- Counting of algae: daily
Cell concentration of each test vessel was determined and recorded daily for 3 days after beginning of the test. Morphological deviations compared to the control cells were recorded at the end of the study. Cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 uL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA).

At the end of the test, thepH of the test media was measured. The test solutions were treated with activated carbon and disposed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Details on results:

Validity criteria:
* Mean biomass increase in the control cultures: 229.0 (required: a factor of at least 16 within the 72h test period)
* Mean coefficient of variation for section-by-section specific growth rates in the control cultures: 6.6% (allowed: up to 35%)
* Coefficient of variation of average specific growth rates during test period in replicate control cultures: 1.3% (allowed: up to 7%)

Reported statistics and error estimates:

The results of the fluorescence readings (RFU, relative fluorescence units) were converted into biomass concentration (Cells/mL) using a calibration line (shown in original report; y = a * x with y = RFU, X = biomass and a= 2310.57 ; R2=0.999). Data were evaluated for normal distribution by Shapiro-Wilk's Test and for homogeneity of variances by Levene's Test on Variance Homogeneity. Student T-test (one-sided) for Homogeneous Variances with Bonferroni-Holm adjustment was applied to find out whether there were significant differences between the growth of the algae in the controls and the algae exposed to the test item concentration. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf, Germany) was used for these calculations.
Statistical data are provided in Annex of the original report.

Treatment (mg test item/L)	% Inhibition of Yield (biomass)	% Inhibition of growth rate
0	0	0
1.0	-6.6	-1.1
3.2	-6.1	-1.1
10	8.3	1.6
32	4.6	0.9
100	9.4	1.8

Conclusions:

An OECD 201 test was conducted under GLP with ammonium thiosulfate. Nominal test concentrations ranged between 1 and 100 mg test item/L. N o statistically significant differences were found between the control and any of the test concentrations. Therefore an unbounded NOEC of >=100 mg ammonium thiosulfate/L was determined (endpoints: biomass yield and growth rate). The EC50 was > 100 mg ammonium thiosulfate/L (endpoints: biomass yield and growth rate). Data can be used for the environmental assessment of ammonium thiosulfate and other thiosulfate compounds (read-across principle).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: Test procedure comparable to standard methods and in accordance with general accepted scientific standards; sufficient documentation.

Justification for type of information:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Growth inhibition test, photosynthetic CO2 fixation inhibition

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: other algae: Scenedesmus brasiliensis; Chlorella vulgaris; Chlamydomonas reinhardii

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20°C

pH:

about 6.2

Nominal and measured concentrations:

0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 10.0 mM (SO3, nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed culture flasks were placed in a temparature controlled water bath
- Aeration: aeration with 5% CO2

GROWTH MEDIUM
- Standard medium used: yes, Kuhl and Lorenzen (1964)
- Detailed composition:
1xE-02 M KNO3
4.5xE-03 M NaH2PO4
0.5xE-01 M Na2HPO4
1xE-03 M MgSO4
1x-04 M CaCl2
2.5xE-05 M FeSO4-EDTA-complex
1xE-06 M H3BO4
1xE-06 M MnSO4
1xE-08 M CuSO4
1xE-08 M NH4Mo7O24


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: at test start
- Photoperiod: continuous
- Light intensity and quality: 3500 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer (optical density was measured), daily
- Other: total protein and photosynthetic 14CO2 Fixation after 96 hours

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

>= 63 - <= 126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Chlorella vulgaris, Scenedesmus brasiliensis, Chlamydononas reinhardtii

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

37.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Chlorella vulgaris, Scenedesmus brasiliensis, Chlamydomonas reinhardtii

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

other: sulfite

Basis for effect:

growth rate

Remarks on result:

other: Chlorella vulgaris

Remarks:

graphical estimate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

other: sulfite

Basis for effect:

growth rate

Remarks on result:

other: Scenedesmus basiliensis

Remarks:

graphical estimate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

other: sulfite

Basis for effect:

growth rate

Remarks on result:

other: Chlamydomonas reinhardii

Growth rate of control: µ = 0.078 h-1 (td = 8.9 h)

EC50 between 0.5 and 1 mM, values taken from a graph, EC50 related to Na2SO3

Conclusions:

Reported data can be used as supportive information for the hazard and effect assessment of sulfite compounds (read-across principle). Based on sulfite concentrations, graphical estimates of EC50 values of 48, 72 and 72 mg/L were derived for Chlorella vulgaris, Scenedesmus basiliensis and Chlamydomonas reinhardtii, respectively.

Description of key information

In a reliable algae growth inhibition study with ammonium thiosulfate,an unbounded NOEC of ≥ 100 mg/L (OECD limit concentration), corresponding to ≥ 54 mg SO32-/L, was found. Hence, a low potential of sulfite/disulfate and thiosulfate substances for algae toxicity is assumed.Due to the observed pH decrease in studies with other sulfites, respective effect concentrations should be considered with caution. Sodium has a low potential for toxicity to algae and cyanobacteria. Based on read-across to sulfite/disulfite/thiosulfate substances and soluble sodium substances and taking into account ubiquitousness of sodium and sulfur and essentiality of sulfur, the potential of disodium disulfite for toxicity to aquatic algae and cyanobacteria is expected to be low. A confirmatory study on the effects of disodium disulfite to Raphidocelis subcapitata according to OECD 201 with appropriate buffering of the test system is currently ongoing.

Additional information

Two studies on the toxicity of disodium disulfite to aquatic algae and cyanobacteria are available (BASF, 1989; Stamm, 1980). In addition, read-across to sulfite/disulfite and thiosulfate substances is applied. Upon contact with water, salts of sulfur oxyacids including disodium disulfite dissociate into sulfur oxyacid anions and respective counterions. Only the properties of the sulfite anion are considered a relevant determinant of environmental toxicity since sodium cations have a low potential for toxicity to aquatic algae and cyanobacteria. Please refer to the endpoint summary for sodium (Na) for further details.

Sulfite/disulfite and thiosulfate substances – Toxicity to aquatic algae and cyanobacteria

A 96-h algal growth inhibition toxicity test was conducted with the green alga S. subspicatus and disodium disulfite, and 72h-EC10 and EC50 values of 33.2 and 43.9 mg test item/L were derived, respectively, corresponding to 28 and 36.8 mg SO32-/L (BASF, 1989). However, the pH of test solutions was significantly lower at concentrations ≥ 62.5 mg/L sodium disulfite, compared to the control and to lower test concentrations. Criteria for algae testing according to OECD 201 and OECD Series 23 ("Guidance document on aquatic toxicity testing of difficult substances and mixtures") regarding the pH of test solutions are as follows: “pH in test vessels should not differ by more than 1.5 from the control medium, otherwise test cultures need to be adapted to the different conditions prior to testing, preferably all test vessels should be adjusted to ± 0.2 at test start. Furthermore, pH should not change by more than 1.5 during the exposure duration”. These criteria were not fulfilled in the study (BASF, 1989), and observed growth inhibition at the level of the EC50 might be due to the pH decrease.

In a supporting study with disodium sulfite (Stamm, 1980), graphical estimates of EC50 values of 48, 72 and 72 mg SO32-/L were derived for Chlorella vulgaris, Scenedesmus basiliensis and Chlamydomonas reinhardtii, respectively. However, since results are not reported in detail and ECx values are graphical estimates, the results are considered supporting only. Furthermore, the pH of test solutions was not measured or not reported, and it cannot be assessed if pH effects occurred.

A reliable study with Pseudokirchneriella subcapitata is available with ammonium thiosulfate (GLP-conform OECD 201 test by Gilberg and Richter, 2010). Nominal test concentrations range from 1 to 100 mg/L, and significant effects were not observed at any of the tested concentrations. Therefore, an unbounded NOEC of ≥ 100 mg ammonium thiosulfate/L and EC50 of > 100 mg ammonium thiosulfate/L were determined (biomass and growth rate) (corresponding to ≥ 54 mg SO32-/L).

In sum,effects observed in the study with disodium disulfite may be due to the pH decrease of test solutions and may not represent the intrinsic toxicity potential of sulfite/disulfite substances.In the reliable study with ammonium thiosulfate, in which the pH remained stable, growth inhibition was not observed up to the tested limit of 100 mg/L (corresponding to 54 mg sulfite/L).In the supporting study by Stamm (1980), information on pH conditions during the test is not available and it is thus not possible to confirm stable conditions. Since derived EC50 values were i) unbounded at the limit concentration of 100 mg/L defined in OECD 201, and/or ii) presumably linked to pH effects and not to the intrinsic toxic properties of sulfite/disulfite substances, it is concluded that sulfite/disulfite substances are not acutely toxic to freshwater algae. However, an algal growth inhibition test on the effects of disodium disulfite according to OECD 201 with appropriate buffering of the test system is planned to confirm this assessment.

Since derived EC10/NOEC values were either unbounded or well above the chronic hazard classification criteria defined in Regulation (EC) No 1272/2008, it is concluded that sulfite/disulfite substances are not chronically toxic to freshwater algae. A confirmatory study on the effects of disodium disulfite to Raphidocelis subcapitata according to OECD 201 with appropriate buffering of the test system is currently ongoing.