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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/2010-12/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
To avoid evaporation of the substance, the Erlenmeyer flasks were closed tightly with glass stoppers. Therefore the pH value decreased by more than 1.5 pH units. This decrease is not regarded to be relevant to the results as all validity criteria were met
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Sampling schedule:
Stock solution: at 0 h
Control: at 72 h
Test concentrations: at 0, 24, 48 and 72 hours
Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
A stock solution was prepared to give the desired series of test concentrations. 90.5 μL of the substance were added to 2 litres of dilution water and treated for 1 h on a magnetic stirrer. Finally undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 μm. The pH was measured to be 7.8.
The concentration of the substance was determined analytically after filtration by HPLC to be 17.9 mg/L resulting in the final concentrations of 0.8, 1.7, 3.7, 8.1 and 17.9 mg/L.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL. Afterwards 0.581 mL of the algal inoculum were added to each replicate resulting in a final cell density of 5000 cells/mL and the vessels were sealed with glass stoppers to avoid evaporation. For each test item concentration and the control 3 replicates were prepared.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Name :
Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
Source :
Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Maintenanceand Acclimatisation :
Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24°C with a maximum fluctuation of +/- 2°C) at a light intensity in the range 60 – 120 μE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
Preparation of pre-cultures :
Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
Test cultures :
The algal inocula for a test were taken from an exponentially growing pre culture and were mixed with the growth medium (annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
1.3°dH (22.5 mg/L CaCO3)
Test temperature:
21 - 24°C
pH:
7.8 - 10.5
The pH value increased by more than 1.5 pH units. This increase is not regarded to be relevant to the results as all validity criteria were met.
Nominal and measured concentrations:
0.8, 1.7, 3.7, 8.1 and 17.9 mg/L
nominal: 0.8 mg/L / measured: 0.541, 0.397, 0.413, 0.343 mg/L at 0, 24, 48, 72 h
nominal: 3.7 mg/L / measured: 2.258, 1.888, 1.992, 1.684 mg/L at 0, 24, 48, 72 h
nominal: 17.9 mg/L / measured: 13.585, 12.065, 11.046, 9.915 mg/L at 0, 24, 48, 72 h
Details on test conditions:
Test vessels :
300 mL Erlenmeyer flasks with glass stoppers test volume: 100 mL
Culturing apparatus :
Light chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks.
Light intensity :
At the average of the test solutions, a light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was used. Light intensity was checked before the start of the study.
Cell density measurements :
Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
Experimental design :
5 test concentrations plus 1 control 3 replicates per concentration, 3 replicates per control; 3 additional replicates each, per test concentration and control for analytical purposes Initial cell density in the test cultures approximately 5000 cells per millilitre.
Test item concentration/s :
0.8, 1.7, 3.7, 8.1 and 17.9 mg/L
Method of administration :
stock solution
Duration of exposure :
72 hours
Criteria of effects :
The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.2 - 3.7
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.96 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Remarks:
(-The factor of biomass parameter 64 > 16; -The mean of the replicate coefficients of variation in the section-by-section growth 25.8 % < 35%; - The coefficient of variation of the mean specific growth rate replicates in the control 0.0 % < 7%.)
Conclusions:
An EC50 of 2.8 and a NOEC of 0.42 mg/L was determined within 72 hours on Desmodesmus subspicatus.
Executive summary:

In order to test acute toxicity to algae of the substance, Desmodesmus subspicatus was exposed to the test solution at 0.8, 1.7, 3.7, 8.1 and 17.9 mg/L nominal concentration of the substance, and blank control solution for a period of 72 h under static conditions. The method used was the OECD Guideline 201 (Alga, Growth Inhibition Test). The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 61.0 to 75.9% of nominal values at 0 hours, from 49.6 to 67.4% of nominal values at 24 hours, from 51.6 to 61.7% of nominal values at 48 hours and from 42.9 to 55.4% at 72 hours. A 72h-EC50 of 2.8 mg/L and a 72h-NOEC of 0.42 mg/L were obtained.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

Description of key information

An EC50 of 2.8 and a NOEC of 0.42 mg/L was determined within 72 hours on Desmodesmus subspicatus.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.8 mg/L
EC10 or NOEC for freshwater algae:
0.42 mg/L

Additional information

Key study:

In order to test acute toxicity to algae of the substance, Desmodesmus subspicatus was exposed to the test solution at 0.8, 1.7, 3.7, 8.1 and 17.9 mg/L nominal concentration of the substance, and blank control solution for a period of 72 h under static conditions. The method used was the OECD Guideline 201 (Alga, Growth Inhibition Test). The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 61.0 to 75.9% of nominal values at 0 hours, from 49.6 to 67.4% of nominal values at 24 hours, from 51.6 to 61.7% of nominal values at 48 hours and from 42.9 to 55.4% at 72 hours. A 72h-EC50 of 2.8 mg/L and a 72h-NOEC of 0.42 mg/L were obtained.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

Supporting study:

The 72-hr toxicity of 2,4-dichlorotoluene to Pseudokirchnerella subcapitata was studied in an open system, a vehicle was used.

The EU-method C3 (1984) was followed.

The 72-h EbC50 was 9.7 mg/L (based on biomass). This value is comparable to the key result.

Reference: EA Japan, 1992

Disregarded study:

The 48-hr toxicity of 2,4 - dichloromethylbenzene to Pseudokirchnerella subcapitata was studied in a closed system. Analytical monitoring was performed and tests with a difference of > 6% of test substance concentrations were repeated. The 48-h EC50 was determined to be 3.53 mg/L based on biomass. Though the test duration of 48 hours is shorter than required for an algae study (72 hours), it is questionable whether longer test duration would have led to a lower EC 50, taking into account that the supporting study below had a duration of 72 hours and yielded an EC50 of 9.7 mg/L. As finally only results for biomass were reported, the study is considered to be invalid.

Reference: Tsai, 2007, 2004

Disregarded study:

The 24-hr toxicity of 2,4-dichlorotoluene toScenedesmus vacuolatuswas studied in a closed system and by using a vehicle. The 24-h EC50 was found to be 4 ng/L. The exposure period was too short (24 h instead of 72h) and no guideline was followed. The study is considered not to be reliable and is therefore disregarded.

Reference: Altenburger, 2004