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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the read-across justification document attached to section 13 in IUCLID.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
500 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
190 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities observed
- Any stimulation of growth found in any treatment: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth.
- Any observations that might cause a difference between measured and nominal values: substance is highly volatile, the arithmetic mean of the measured values at t=0 and t=72h are reported
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h ErC50 = 0.90 mg/L
- 72h EbC50 = 0.40 mg/L
- 72h EyC50 = 0.32 mg/L

ANALYTICAL RESULTS

In the limit test (experiment 1) the measured concentrations showed medium correlation to the nominal concentrations (77% and 70% of nominal concentration were measured). The recovery rate after 72 h was 91% of the start concentration. Therefore, the arithmetic mean of measured concentration (at t = 0 and 72 h) was used for the determination of the results.

In experiment 2, the measured concentrations ranged from 5 - 70% of the nominal concentratio at t = 0 h and from 4 - 64% at t = 72 h. The recovery rate after 72 h was very good. The measured value in treatment 3200 mg/L (nominal) was much too low, probably because of insufficient homogenisation (recovery after 72 h = 369%). In spite of different nominal concentrations, the measured concentrations in treatments above 320 mg/L were in a similar range. It is assumed that this effect is caused by the limited volatility of the test item from the aqueous medium into the headspace. The measured concentrations of the nominal concentration 10000 mg/L were not considered valid and were not used for evaluation.

BIOLOGICAL RESULTS

In experiment 1 (limit test) no inhibition of algal growth was detected. The nominal concentration of 100 mg/L (mean of measured concentrations = 74 mg/L) showed enhancement of algal growth. But the measured concentration was too low, so that no conclusion could be drawn about the effect on algal growth at the nominal limit concentration of 100 mg/L test item. Therefore, a second experiment was conducted, using 5 treatments in a geometric series.

In experiment 2, nearly no algal growth was observed in treatments above 320 mg/L (nominal). The nominal concentration of 320 mg/L (mean of measured concentrations = 180 mg/L) showed strong inhibition. Therefore, the LOEC was stated as range between 74 and 180 mg/L.

Conclusions:
Since no data on the acute toxicity of CAS 540-54-5 to algae are available a read-across from a supporting substance was conducted. Please refer to the read-across justification document attached to section 13 in IUCLID. Based on the available data an ErC50 (72 h) of 500 mg/L (meas., arithm. mean) and a
NOErC (72 h) of 74 mg/L (meas., arithm. mean).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 - 28 May (exp. 1) and 16 - 19 Jul (exp. 2) 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to the read-across justification document attached to section 13 in IUCLID.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
reliability scoring based on 2006 guideline
Deviations:
yes
Remarks:
; The pH varied by > 1.5 units in some experiments, the temperature exceeded 24 ºC in some experiments, deemed to be uncritical.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008 followed, reliability scoring based on 2009 guideline.
Deviations:
yes
Remarks:
; The pH varied by > 1.5 units in some experiments, the temperature exceeded 24 ºC in some experiments, deemed to be uncritical. Stock solution II was prepared according to the OECD guideline 201.
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L at t = 0 and t = 72 h (experiment 1); 320, 1000, 3200, 10000 mg/L at t = 0 and t = 72 h (experiment 2)
- Sampling method: A sample quantity of 1 mL test solution was taken with a syringe and transferred into a GC vial. The vials were immediately closed. After 15 minutes at 20 °C, a sample volume of 50 µL was taken from the headspace and measured via GC/FID.
- Sample storage conditions before analysis: none
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the volatility of the test item, the study was performed in a closed system. The test vessels were filled with test medium (deionised water, nutrient solution and algal pre-culture), and the nominal load of test item was directly injected into the vessels with a suitable syringe. After addition of the test item, the vessels were immediately closed.
- Differential loading: The nominal load of test item (15 µL experiment 1; 48 / 150 / 480 7 1500 µL experiment 2) was directly injected into the vessels with a suitable syringe or pipette.
- Controls: Yes, 6 replicates (deionised water, nutrient medium and alga)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular freshwater green alga
- Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen, Mar 2010)
- Age of inoculum (at test initiation): Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 96 hours.
- Method of cultivation: The algae are kept as stock culture on solid agar at 8 °C. The pre-culture was prepared from an aliquot of this stock culture.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
24 - 25 ºC (experiment 1)
25 - 26 ºC (experiment 2)
24 - 30 ºC (reference study)
pH:
7.0 - 8.4 (experiment 1)
control: 7.0 - 9.2; 320 mg/L: 7.0 - 6.9; 1000 mg/L: 7.0 - 7.2; 3200 mg/L: 7.0 - 5.9; 10000 mg/L: 7.0 - 8.4 (experiment 2)
Nominal and measured concentrations:
nominal: control, 100 mg/L (experiment 1); control, 320, 1000, 3200, 10000 mg/L (experiment 2)
mean measured: < LOD, 74 mg/L (experiment 1); < LOD, 178, 670, 641, 449 mg/L (experiment 2)
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type: closed
- Material, size, headspace, fill volume: glass; completely filled with test medium, total volume 130 mL
- Initial cells density: 14708 cells/mL (experiment 1), 7354 cells/mL (experiment 2)
- Control end cells density: 275775 cells/mL (experiment 1), 1161932 cells/mL (experiment 2)
- No. of vessels per concentration (replicates): 6 replicates (experiment 1), 3 replicates (experiment 2)
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, to avoid a high pH increase, 700 mL deionised water was mixed with 6 mL sodium hydrogencarbonate solution (50 g/L). Then, pH was adjusted to 7.0 (± 0.2) with HCl.
- Photoperiod: continuous
- Light intensity and quality: 5900 - 6900 lux (experiment 1); 4900 - 5100 lux (experiment 2)
- Other: During the test, the test vessels were shaken on an orbital shaker in the light incubator.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer.

TEST CONCENTRATIONS
Because of the weak correlation between nominal (100 mg/L) and measured concentrations (74 mg/L) in experiment 1, additional treatments ranging from 320 to 10000 mg/L (nominal concentration) were tested in a second experiment.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
500 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
190 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 74 - <= 180 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth, 180 mg/L showed strong inhibition, therefore, LOEC range between 74 and 180 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities observed
- Any stimulation of growth found in any treatment: 74 mg/L (nominal concentration 100 mg/L) showed enhancement of algal growth.
- Any observations that might cause a difference between measured and nominal values: substance is highly volatile, the arithmetic mean of the measured values at t=0 and t=72h are reported
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h ErC50 = 0.90 mg/L
- 72h EbC50 = 0.40 mg/L
- 72h EyC50 = 0.32 mg/L

ANALYTICAL RESULTS

In the limit test (experiment 1) the measured concentrations showed medium correlation to the nominal concentrations (77% and 70% of nominal concentration were measured). The recovery rate after 72 h was 91% of the start concentration. Therefore, the arithmetic mean of measured concentration (at t = 0 and 72 h) was used for the determination of the results.

In experiment 2, the measured concentrations ranged from 5 - 70% of the nominal concentratio at t = 0 h and from 4 - 64% at t = 72 h. The recovery rate after 72 h was very good. The measured value in treatment 3200 mg/L (nominal) was much too low, probably because of insufficient homogenisation (recovery after 72 h = 369%). In spite of different nominal concentrations, the measured concentrations in treatments above 320 mg/L were in a similar range. It is assumed that this effect is caused by the limited volatility of the test item from the aqueous medium into the headspace. The measured concentrations of the nominal concentration 10000 mg/L were not considered valid and were not used for evaluation.

BIOLOGICAL RESULTS

In experiment 1 (limit test) no inhibition of algal growth was detected. The nominal concentration of 100 mg/L (mean of measured concentrations = 74 mg/L) showed enhancement of algal growth. But the measured concentration was too low, so that no conclusion could be drawn about the effect on algal growth at the nominal limit concentration of 100 mg/L test item. Therefore, a second experiment was conducted, using 5 treatments in a geometric series.

In experiment 2, nearly no algal growth was observed in treatments above 320 mg/L (nominal). The nominal concentration of 320 mg/L (mean of measured concentrations = 180 mg/L) showed strong inhibition. Therefore, the LOEC was stated as range between 74 and 180 mg/L.

Description of key information

ErC50 (72 h): 500 mg/L (arithmetic mean measured, D. subspicatus, OECD 201)

NOErC (72 h): 74 mg/L (arithmetic mean measured, D. subspicatus, OECD 201)

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
500 mg/L
EC10, LC10 or NOEC for freshwater algae:
74 mg/L

Additional information

As no study is available for assessing the toxicity of the substance on algae, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 Grouping of substances, a read-across to the structurally related 2 -Chloropropane (CAS 75-29-6) was applied. The read across is justified due to the structural similarity of the analogue substances and similar physico-chemical properties resulting in a similar environmental fate and ecotoxicity profile. Further justification is given in the analogue justification attached to section 13 in IUCLID.

There is one GLP study available investigating the effects microalgae of a structural analog of the test substance on the growth of freshwater microalgae according to the OECD guideline 201.

In this study, two static experiments were performed exposing the green alga Desmodesmus subspicatus to the test substance for 72 h under controlled conditions. Both tests were performed in a closed system due to the volatility of the test substance. The first experiment was performed as a limit test using the nominal concentration 100 mg/L. The second experiment was conducted with five treatments in a geometric series with nominal concentrations ranging from 320 – 10000 mg/L. The second study was considered necessary because no inhibition was observed at 100 mg/L and no conclusion could be drawn about affection of the alga at this limit concentration. The test substance was chemically analyzed at the start (t = 0 h) and end (t = 72 h) of the experiment. To determine the growth rate, the area under the growth curve as well as the yield, the cell concentration of each replicate was determined by measuring the absorption at 440 nm every 24 h with a spectral photometer.

In the first experiment, the recovery after 72 h was 70% of the start concentration. Therefore, the determination of the results was based on the mean measured concentration of t = 0 and t = 72 h. This resulted in a mean measured concentration of 74 mg/L for the nominal treatment 100 mg/L. In the second experiment, the recovery rate after 72 h, with respect to the measured initial concentration at t = 0 h, was very good. However, the measured concentration of the nominal concentration of 10000 mg/L was not considered valid and omitted.

The nominal concentration 100 mg/L (mean measured = 74 mg/L) showed enhancement of algal growth. The nominal concentration 320 mg/L (mean measured = 180 mg/L) showed strong inhibition. In treatments above 320 mg/L (nominal), nearly no algal growth was observed. Therefore, the LOEC was stated as a range between 74 and 180 mg/L (arithmetic mean measured). The derived ErC50 (72 h) was 500 mg/L (arithmetic mean measured) and the NOErC (72 h) was 74 mg/L (arithmetic mean measured).