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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Remarks:
Constituent of the reaction mass
Adequacy of study:
key study
Study period:
From 26 November 1997 to 05 September 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study scored as Klimisch 2 because not performed on the registered substance itself but on one of its constituent

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
only 4 laoding rates were tested without justification within the report. As EC50 cannot be calculated with the data obtained, there is no incidence of using only 4 loading rates.
Principles of method if other than guideline:
This study is divided in two parts:
- firstly, the report contains a classical EU C.3 study measuring the toxicity of dicerium trisulphide to algal growth.
- secondly, the report contains a technical notice summarizing assays aiming at determining the interaction between dicerium trisulphide and a nutrient salt (phosphate) on algal growth. In these assays, algae were exposed to dicerium trisulphide in media complemented or not with a concentrated solution of nutrient salts.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Dicerium trisulphide
EC Number:
234-603-7
EC Name:
Dicerium trisulphide
Cas Number:
12014-93-6
Molecular formula:
Ce2S3
IUPAC Name:
Dicerium trisulphide
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: Sulfure de cérium

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
STUDY PART 1 - EU C.3:

- In this study, cerium was analysed by Inductively Coupled Plasma - Mass Spectrometry (ICP - MS).
- Concentrations: only the control and the 100 mg/L nominal loading rate treatments were sampled for analysis.
- Sampling method: 50 mL plastic syringe or 10 mL plastic pipette were used to collect the different samples. Different samples were taken:
* Samples taken during the preparation of the test solution; that is after 6 days (144 hours) of stirring.
* Samples taken at the end of the preparation of the test solution; that is after 7 days (168 hours) of stirring. These samples were thus collected just before algae exposure. Therefore, the corresponding analytical measurements can be assimilated to t = 0.
* Samples taken after 72 hours of algae exposure (t = 72). It has to be noticed that this 72-hour solution was kept in the same conditions than the solution used for exposure but without the inoculum.
- Sample storage conditions before analysis: in plastic containers at about 4-5°C.

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STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE:

- In these assays, cerium and phosphate were analysed by ICP - MS and Ionic Chromatography, respectively.
- Sampling method / Concentrations: Samples for analysis were collected in the control and the 100 mg/L nominal loading rate treatments (no data about the material used to collect samples). Analyses were performed on samples submitted to different conditions:
* Samples were analysed directly.
* Samples were analysed after a 30-min centrifugation.
* Samples were analysed after phosphate complementation and 24-hour stirring.
* Samples were analysed after phosphate complementation, 24-hour stirring and 30-min centrifugation.
- Sample storage conditions before analysis: in plastic containers at about 4°C.

Test solutions

Vehicle:
no
Details on test solutions:
STUDY PART 1 - EU C.3:

- Method of preparation and application of test solution: The test substance and 1800 g of the test medium were introduced in 2-liter polypropylene beakers in the respective proportions of 0, 1, 10, 32 and 100 mg per 1000 g. The contents of the beakers were magnetically stirred (60-mm long cylindrical magnetic bar) during 7 days (168 hours) at about 20°C in such a manner that the vortex almost produced air bubbles in the solution. Evaporation was measured and compensated by double distilled water addition. After 168 hours stirring, the contents of the beakers were filtrated with a sight pressure on Millipore membrane HAWP04700 of 0.45 µm porosity. The solutions obtained were used as the test solutions and are referred as Water Soluble Fractions (WSF).
- Differential loading: The 4 loading rates were prepared individually (and not by dilution from a stock solution).
- Controls: Blank (test water without addition of the test item)
- Evidence of undissolved material: No data

-------------------------------

STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE:

- Method of preparation and application of test solution: The procedure with respect to stirring (material, duration), filtration, etc., was exactly the same as for the classical EU C.3 study, except that here, different loading rates were tested in 5 different assays:
* Assay 1: WSF prepared at a loading rate of 100 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2%, 1.8%, 1% WSF.
* Assay 2: WSF prepared at a loading rate of 32 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2% WSF.
* Assay 3: WSF prepared at a loading rate of 10 mg/L and test of 100%, 56%, 32%, 18%, 10% WSF.
* Assay 4: WSF prepared at a loading rate of 0 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2%, 1.8%, 1% WSF.
* Assay 5: WSF prepared at loading rates of 1, 10, 32 and 100 mg/L. These four different 100% WSF were complemented with a concentrated solution of nutrient salts. The added amount of nutrients was made in order to double the theoretical concentrations of the nutrients in the WSFs.
- Differential loading: The different loading rates were prepared individually (and not by dilution from a stock solution).
- Controls: Blank (test water without addition of the test item)
- Evidence of undissolved material: No data

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4
- Source: Stored and cultivated in the laboratory of ECOTOXICOLOGY CRIT/Decines
- Age of inoculum (at test initiation): Algae were taken from an exponentially growing pre-culture, which was set up four days prior to the test into the same test medium.
- Method of cultivation: No data
- Any deformed or abnormal cells observed: morphological control of the strain was done before the preparation of the pre-inoculum

ACCLIMATION
- Acclimation period: No

Study design

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

Test conditions

Test temperature:
STUDY PART 1 - EU C.3: Between 22.9 and 23.2°C

STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE: 22.9 and 23.2°C
pH:
STUDY PART 1 - EU C.3: Between 7.4 and 7.8

STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE: No data
Nominal and measured concentrations:
STUDY PART 1 - EU C.3:
- Nominal loading rates: 0 - 1 - 10 - 32 and 100 mg/L
- Mesured Cerium concentrations: Analytical measurements were performed only in the control and 100 mg/L loading rate treatments:
* Control samples: < 0.2 µg/L at each time points (i.e. during the preparation of the test solutions on the day 6th and 7th, thus after 144h and 168h of stirring, and after 72 hours of algae exposure)
* 100 mg/L loading rate samples:
> During the preparation of the test solutions on the 6th day (thus after 144 hours of stirring): 9.7 µg/L
> During the preparation of the test solutions on the 7th day (thus after 168 hours of stirring): 8.5 µg/L (t = 0)
> After 72 hours of algae exposure: 7.8 µg/L (t = 72)

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STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE:
- Nominal loading rates: 0 - 1 - 10 - 32 and/or 100 mg/L depending on the assays
- Mesured Cerium concentrations: Analytical measurements were performed only in the control and 100 mg/L loading rate treatments:
* Control samples: < 0.2 µg/L.
* 100 mg/L loading rate samples: Analyses were performed on samples submitted to different conditions:
> Samples analysed directly: 7 µg/L
> Samples analysed after a 30-min centrifugation: 4.7 to 8 µg/L
> Samples analysed after phosphate complementation and 24-hour stirring: 3 µg/L
> Samples analysed after phosphate complementation, 24-hour stirring and 30-min centrifugation: < 0.2 µg/L
Details on test conditions:
STUDY PART 1 - EU C.3:

TEST SYSTEM
- Test vessel: 180 mL non-reusable plastic (polypropylene) containers (Before utilisation, the container were soaked in double distilled water during 24 hours).
- Type: open
- Material, size, headspace, fill volume: vessels filled with 30 mL of test culture, 2% (of total culture volume) of inoculum
- Aeration: no, but algal suspensions were continuously shaken at 140 strockes per minute
- Initial cells density: 10E4 cells per mL
- Control end cells density (OD-665nm-1cm): 267.8 (relation used between optical density OD and number of algal cells per mL (N): an OD (665nm-5cm) = 0.00066 N)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium with a very small deviation (FeCl3, 6H2O: 0.08 mg/L instead of 0.064 mg/L and H3BO3: 0.18 mg/L instead of 0.185 mg/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water. The test medium and the water were sterile. Sterilizations were carried out by autoclaving or by filtration on a Sartorius type 11107 membrane of 0.2 µm porosity
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: No data available
- Culture medium different from test medium: No data available
- Intervals of water quality measurement:
* The temperature was continuously measured in a container filled with a water volume identical to the test culture volume, and placed in the same conditions.
* The pH of each test culture was measured at the beginning and after 72 hours of incubation.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuously illuminated
- Light intensity and quality: The light (white light) had a continuous spectrum between 400 and 700 nm, and an intensity between 6000 and 10000 lux (PHILIPS TLD 58 W 86)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The optical density (OD) was measured at the beginning and after 24, 48, and 72 hours of incubation. Two growth parameters were then calculated from these OD measurements:
* Area under the growth curve (= integral biomass).
* Growth rate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 (100 - 32 - 10 - 1 mg/L nominal loading rates) knowing that a higher factor was used between the two lowest loading rates.
- Justification for using less concentrations than requested by guideline: only 4 loading rates were tested without justification within the report. As EC50 cannot be calculated with the data obtained, there is no incidence of using only 4 loading rates.
- Range finding study: No

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STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE:

TEST SYSTEM
- Test vessel: 180 mL single-use polypropylene containers (Before utilisation, the container were soaked in double distilled water during 24 hours).
- Aeration: no, but algal suspensions were continuously shaken at 140 strockes per minute
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
No further data

GROWTH MEDIUM
- Standard medium used: yes, OECD medium with a very small deviation (FeCl3, 6H2O: 0.08 mg/L instead of 0.064 mg/L and H3BO3: 0.18 mg/L instead of 0.185 mg/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water. The test medium and the water were sterile. Sterilizations were carried out by autoclaving or by filtration on a Sartorius type 11107 membrane of 0.2 µm porosity
- Intervals of water quality measurement:
* The temperature was continuously measured in a container filled with a water volume identical to the test culture volume, and placed in the same conditions.
No further data.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuously illuminated
- Light intensity and quality: The light (white light) had a continuous spectrum between 400 and 700 nm, and an intensity between 6000 and 10000 lux (PHILIPS TLD 58 W 86)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The optical density (OD) was measured after 72 hours of incubation.

No further data (less experimental details are available in the report for study part 2 as compared to study part 1).
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)

Results and discussion

Effect concentrations
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks on result:
other: Cannot be calculated (see below)
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- 72h-EbC50 (Raphidocelis subcapitata): 0.62 mg/L.
Reported statistics and error estimates:
STUDY PART 1 - EU C.3: EC50 and their confidence limits cannot be calculated with the data obtained. Therefore, no statistics was performed.

STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE: Biomass results obtained from optical density (OD) measurements were provided, but not statistical analysis is available in the report.

Any other information on results incl. tables

STUDY PART 1 - EU C.3:

For integral biomass, the following inhibition percentages were obtained:

100 mg/L loading rate: 82%

32 mg/L loading rate: 83%

10 mg/L loading rate: 80%

1 mg/L loading rate: 2%

For growth rate, the following inhibition percentages were obtained:

100 mg/L loading rate: 62%

32 mg/L loading rate: 66%

10 mg/L loading rate: 59%

1 mg/L loading rate: 0%

EC50 and their confidence limits cannot be calculated with the data obtained.

STUDY PART 2 - INTERACTION BETWEEN DICERIUM TRISULPHIDE AND PHOSPHATE:

Assay 1:

Reminder on the tested treatments:

WSF prepared at a loading rate of 100 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2%, 1.8%, 1% WSF.

Obtained results:

100 %WSF: 89% inhibition final biomass

56% WSF: 22% inhibition final biomass

32% WSF: 11% inhibition final biomass

18% WSF: 8% inhibition final biomass

10% WSF: 7% inhibition final biomass

5.6% WSF: 5% inhibition final biomass

3.2% WSF: 4% inhibition final biomass

1.8% WSF: 5% inhibition final biomass

1% WSF: 8% inhibition final biomass

 

Assay 2:

Reminder on the tested treatments:

WSF prepared at a loading rate of 32 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2% WSF.

Obtained results:

100 %WSF: 90% inhibition final biomass

56% WSF: 21% inhibition final biomass

32% WSF: 9% inhibition final biomass

18% WSF: 8% inhibition final biomass

10% WSF: 6% inhibition final biomass

5.6% WSF: 8% inhibition final biomass

3.2% WSF: 5% inhibition final biomass

 

Assay 3:

Reminder on the tested treatments:

WSF prepared at a loading rate of 10 mg/L and test of 100%, 56%, 32%, 18%, 10% WSF.

Obtained results:

100 %WSF: 88% inhibition final biomass

56% WSF: 20% inhibition final biomass

32% WSF: 9% inhibition final biomass

18% WSF: 59% inhibition final biomass (probably an outlier)

10% WSF: 6% inhibition final biomass

 

Assay 4:

Reminder on the tested treatments:

WSF prepared at a loading rate of 0 mg/L and test of 100%, 56%, 32%, 18%, 10%, 5.6%, 3.2%, 1.8%, 1% WSF.

Obtained results:

Previous inhibition percentages were calculated from the biomass obtained in this assay performed without test item.

 

Assay 5:

Reminder on the tested treatments:

WSF prepared at loading rates of 1, 10, 32 and 100 mg/L. These four different 100% WSF were complemented with a concentrated solution of nutrient salts. The added amount of nutrients was made in order to double the theoretical concentrations of the nutrients in the WSFs.

Obtained results:

100 mg/L loading rate: 12% stimulation final biomass

32 mg/L loading rate: 9% stimulation final biomass

10 mg/L loading rate: 9% stimulation final biomass

1 mg/L loading rate: 18% stimulation final biomass

Conclusions:

- Assays 1 to 3 showed that the WSF of dicerium trisulphide had inhibitory properties, but that the growth inhibition were not related to the initial loading rate of the WSF.

- Assay 5 showed that the addition of nutrient salts to the WSFs removed the inhibitory properties of these solutions.

- The analytical study showed that phosphate is under the limit of detection in the 100 mg/L WSF. This study also showed that the addition of nutrient salts induced the disappearance of the soluble cerium. There is probably an interaction between phosphates and cerium, inducing the precipitation of phosphates during the WSFs preparation (as cerium is in excess), and the precipitation of cerium when nutrient salts are added to the WSFs (as phosphate is in excess). The possible interaction between the test substance and the assay medium prevents for making any conclusion regarding the inhibitory effect of dicerium trisulphide on the algal growth. As the growth of algae is not possible without phosphate, the growth inhibition observed in study part 1 (EU C.3) may be explained by the very low level of phosphate in the test solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The cell density in the control cultures increased by a factor of 160 within 72 hours. The pH of the cultures did not deviate by more than 1 unit during the test.
Conclusions:
This study demonstrates that there is probably an interaction between phosphates and cerium, inducing the precipitation of phosphates during the test solution preparation (as cerium is in excess), and the precipitation of cerium when nutrient salts are added to the test medium (as phosphate is in excess). The possible interaction between the test substance and the test medium prevents for making any conclusion regarding the inhibitory effect of dicerium trisulphide on the algal growth. As the growth of algae is not possible without phosphate, the growth inhibition observed without complementation of the medium with nutrient salts may be explained by the very low level of phosphate in the test solutions. This hypothesis is confirmed by the fact that the addition of nutrient salts removes the inhibitory properties of dicerium trisulphide.
Executive summary:

This study was performed to determine the effect of dicerium trisulphide on the growth of the green alga. The toxicity of dicerium trisulphide to Pseudokirchnerella subcapitata was studied under static conditions according to the EU Commission Directive 92/69/EEC, Part C.3 (1992). This study is divided in two parts:

- firstly, the report contains a classical EU C.3 study measuring the toxicity of dicerium trisulphide to algal growth.

- secondly, the report contains a technical notice summarizing assays aiming at determining the interaction between dicerium trisulphide and a nutrient salt (phosphate) on algal growth. In these assays, algae were exposed to dicerium trisulphide in media complemented or not with a concentrated solution of nutrient salts.

This study demonstrates that there is probably an interaction between phosphates and cerium, inducing the precipitation of phosphates during the test solution preparation (as cerium is in excess), and the precipitation of cerium when nutrient salts are added to the test medium (as phosphate is in excess). The possible interaction between the test substance and the test medium prevents for making any conclusion regarding the inhibitory effect of dicerium trisulphide on the algal growth. As the growth of algae is not possible without phosphate, the growth inhibition observed without complementation of the medium with nutrient salts may be explained by the very low level of phosphate in the test solutions. This hypothesis is confirmed by the fact that the addition of nutrient salts removes the inhibitory properties of dicerium trisulphide.

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