Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 24 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
EC Number:
222-468-7
EC Name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
Cas Number:
3483-12-3
Molecular formula:
C4H10O2S2
IUPAC Name:
1,4-disulfanylbutane-2,3-diol
Specific details on test material used for the study:
Batch no.: 44606300 / 50774
Storage conditions: 2–8°C, under nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test media including the control at the start of the test (without algae) and after 24, 48 and 72 hours of exposure (containing algae). For sampling at the start of the test the samples were taken from the freshly prepared test solutions as well as from the control. For all subsequent sampling time points aliquots from the respective replicates were pooled per treatment before sampling. These samples were also centrifuged at 4500 g for 10 min to separate the algae.
All samples were diluted with acetonitrile containing 1.0% conc. phosphoric acid to obtain a composition of sample:acetonitrile (9:1; v:v) containing 0.1% conc. phosphoric acid. Subsequently, the samples were stored deep-frozen (at about -20 °C) until analysis. In non-GLP preliminary experiments, the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test water, test item and algae (three replicates per test concentration)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item is soluble, the test solutions were set up by respective dilutions of a suitable stock solution, which was prepared by adding 100 mg/l of the test item to test water in sterile glassware and stirred until dissolution. The respective dilutions of this stock solution were then prepared with test water and sterile glassware. 100 ml of each test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
For the exposure, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per minute. The test flasks were repositioned daily during the exposure.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test organism: Raphidocelis subcapitata (SAG Nr. 61.81), supplied by the SAG Culture Collection of Algae at Göttingen University, 37073 Göttingen, Germany.
- Culture: 250 ml flask containing 100 ml of OECD medium inoculated with an exponentially growing preculture of Raphidocelis subcapitata.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation, source: Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).
- Temperature: 21–24 °C maintained at ±2 °C in a thermo-controlled room.
- Control of sensitivity: For evaluation of the algal quality and the test procedure, potassium dichromate is tested as a positive control twice a year.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
he calculated water hardness of the test water is 24.2 mg/l as CaCO3.
Test temperature:
21–24 °C maintained at ±2 °C in a thermo-controlled room.
pH:
7.0 - 8.0
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
The concentrations of the test item (DTT) as well as its oxidation product (ox. DTT) in the test media were analysed by HPLC with UV detection in samples from the test start and from 24, 48 and 72 hours of exposure.
The test item was correctly dosed at test start, but degradation set in very quickly, which led to reduced recovery rates at the lowest two test concentrations. After 24 hours of exposure, DTT could only be found in the highest test concentration, whereas it has been almost completely oxidised at all lower concentrations. The remaining DTT in the highest test concentration degraded over the subsequent 24 hours. At test end, 74-84% of the nominal concentration were found as oxidised DTT. The presence of additional peaks in the HPLC chromatograms of aged samples suggests further degradation.
Based on the results of the non-GLP preliminary tests (see section 4.6), the test item itself as well as its degradation products have been shown to exhibit toxic properties. As it is not possible to distinguish the exact proportions to which each species contributed and also taking into account combined toxicity effects, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers.
- Test medium: Reconstituted water (OECD medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity and homogeneity: Continuous, 4440 – 8880 lux ± max. 15% variation. The test flasks were repositioned daily during the exposure
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: Approximately 5’000 cells/ml; all test media contained the same initial density of algal cells.

RANGE FINDING STUDY
The test item is known to oxidise in the presence of oxygen, especially at pH values =7.5. Non-GLP preliminary stability tests in test water at about pH 7.0 already showed rapid transformation of the test item molecule (DTT) as well as the formation of a degradation product (oxidised DTT).
Together with the sponsor it was decided to perform two non-GLP range finding tests (three replicates for the control and two replicates per test concentration) to determine, which setup led to higher toxicity. One test was performed with nominal concentrations of 1, 10 and 100 mg/l of the test item itself and one with 100%, 10% and 1% (vol.) of a 100 mg/l test item solution, which had been stirred for 96 hours to let the test item completely oxidise. The test water applied in both tests was set to about pH 7.0.

EFFECT PARAMETERS MEASURED
- Biomass: Algal biomass is determined at 24, 48 and 72 hours of exposure using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). To this end, aliquots of 5 ml are removed from each test flask. At test start (0 hours) the nominal value is applied.
Reference substance (positive control):
yes
Remarks:
Control of sensitivity: Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
24.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
AVERAGE SPECIFIC GROWTH RATE
With respect to the endpoint average specific growth rate the following effects were observed compared to the control: 86% at 100 mg/l, 66% at 32.0 mg/l and 15% at 10.0 mg/l. No significant adverse effects were observed at the lowest two test concentrations.
The 72-hour EC50 based on average specific growth rate of 1,4-Dithiothreitol to Raphidocelis subcapitata was calculated to be 24.3 mg/l (95% confidence interval: 21.7–27.2 mg/l). The EC10 was calculated to be 6.77 mg/l (95% confidence interval: 5.17–8.30 mg/l). These ECx were computed using a linear regression.
The LOEC with respect to average specific growth rate was determined to be 10.0 mg/l according to a Williams’ multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 3.20 mg/l.

YIELD
With respect to the endpoint yield, the following effects were observed compared to the control: 100% at 100 mg/l, 98% at 32.0 mg/l, 57% at 10.0 mg/l and 10% at 3.20 mg/l. No significant effects were observed at the lowest test concentration.
The 72-hour EC50 based on yield of 1,4-Dithiothreitol to Raphidocelis subcapitata was calculated to be 8.66 mg/l (95% confidence interval: 8.23–9.11 mg/l). The EC10 was calculated to be 3.36 mg/l (95% confidence interval: 2.96–3.74 mg/l). These ECx were computed using a linear regression.
The LOEC with respect to yield was determined to be 3.20 mg/L according to a Williams’ multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 1.00 mg/l.

OBSERVATIONS AND ENVIRONMENTAL CONDITIONS
The microscopic examination of the algal cells at the end of the test showed no abnormal appearance of the algae growing at a nominal concentration of 10.0 mg/l. The appearance of the algal cells was not affected by the test item up to at least this concentration. The appearance of the algal cells at the highest two test concentrations of 100 and 32.0 mg/l nominal could not be examined due to the low number of algal cells at the end of the exposure.
No remarkable observations were made concerning the appearance of the test media.
The environmental conditions (pH, light, temperature) are summarised in Table 12. The pH value in the control replicates changed by 1.0 units. The light intensity was maintained at 4799 lux ± 7.5%. The temperature was 22 °C over the whole exposure period. All values were within the guideline ranges.
Reported statistics and error estimates:
Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Pro.

Any other information on results incl. tables

Validity of the test:

 Validity criterion

(applies to control cultures)

 Required value  Achieved value
 Average specific growth rate [d-1]  >=0.92 1.83 

CV of average specific growth rate

over 3 days [%]

 <=7  0.6

 CV of section-by-section specific

growth rates [%]

 <=35

 4.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See table in section "any other information on results incl. tables".
Conclusions:
The acute toxicity of 1,4-Dithiothreitol (CAS no. 3483-12-3) to the green alga Raphidocelis subcapitata was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of 1,4-Dithiothreitol to the green alga Raphidocelis subcapitata was estimated to be 24.3 mg/l. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 3.2 mg/l. The results of the test can be considered reliable without restriction.
Executive summary:

The growth inhibitory effects of 1,4-Dithiothreitol (CAS no. 3483-12-3) to the green alga Raphidocelis subcapitata were investigated according to the OECD guideline 201 over a period of 72 hours.

The test item 1,4-Dithiothreitol is solid, 99.3% m/m pure and soluble in test water up to at least 100 mg/l. Consequently, the test solutions were prepared by respective dilutions of a stock solution. The nominal test item concentrations were 100, 32.0, 10.0, 3.20 and 1.00 mg/l.

The test item quantification was performed by HPLC with UV detection and not only included the analysis of the test item (DTT) itself, but also of its main degradation product (ox. DTT). Both were analysed in samples from the test start and from 24, 48 and 72 hours of exposure. The test item was correctly dosed at test start, but degradation set in very quickly. At test end, 74-84% of the nominal concentration were found as ox. DTT. The presence of additional peaks in the HPLC chromatograms of aged samples suggests further degradation. The test item itself as well as its degradation products have been shown to exhibit toxic properties in non-GLP preliminary tests. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item, as it is not possible to distinguish the exact proportions to which each species contributed and also taking into account combined toxicity effects.

With respect to the endpoint average specific growth rate the following effects were observed compared to the control: 86% at 100 mg/l, 66% at 32.0 mg/l and 15% at 10.0 mg/l. No significant adverse effects were observed at the lowest two test concentrations.

With respect to the endpoint yield, the following effects were observed compared to the control: 100% at 100 mg/l, 98% at 32.0 mg/l, 57% at 10.0 mg/l and 10% at 3.20 mg/l. No significant effects were observed at the lowest test concentration.

The results of the average specific growth rate and yield inhibition of the test item to the green alga Raphidocelis subcapitata are summarised in the following table based the nominal concentrations of the test item:

   Parameter (0-72 h)   Growth rate [mg/l]  Yield [mg/l]
EC50  24.3  8.66
EC10   6.77  3.36
 LOEC  10.0  3.2
 NOEC  3.2  1.0

The 72-h EC50 value based on average specific growth rate of 1,4-Dithiothreitol (CAS no. 3483-12-3) on the green alga Raphidocelis subcapitata was 24.3 mg/l and the EC10 was 6.77 mg/l based on the nominal concentration of the test item.

All validity criteria were fulfilled.

If table necessary, otherwise delete it!

cl: confidence limit