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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
For the analysis of the actual test item concentrations the following samples were taken:
- just before the start of the test: duplicate samples from each test medium (without algae) and duplicate samples from the control (without algae)
- after 72 hours (stability samples): duplicate samples from each test medium (without algae) and duplicate samples from the control (without algae)

For the 72-hour stability samples addifional flasks with adequate volumes of the freshly prepared test media of all test concentrafions and the control were incubated under the same condifions as in the actual test but without algae.

All the samples as listed below were analyzed immediately after sampling without prior storage.
The concentrafions of the test item were analyzed in all test medium samples from the first sampling time (0 hours). At the end of the test (72 hours) only the samples of the undiluted filtrate were analyzed. The 72-hour samples of the dilutions 1:2, 1:4, 1:8 and 1:16 were not analyzed, since these concentrations were below the 72-hour NOEC determined in this test. From the control samples only one of the duplicate samples was analyzed from each of both sampling times.
Vehicle:
no
Details on test solutions:
According to the results of a pre-experiment (without GLP) the test item was not soluble at a concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared. Therefore, a filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilufions 1:2, 1:4, 1:8 and 1:16 were tested.
Additionally, a control was tested in parallel (test water without test item). The preparation of the test media was performed as far as possible in the dark to avoid photolytic degradafion of the test item during handling.
A supersaturated dispersion with a nominal concentration of 100 mg/L (= loading rate) was prepared by weighing 449 mg of the test item into 4500 mL test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible by ultrasonic treatment for 15 minutes and by intense sfirring for 3 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion. The stirring period of 3 hours was chosen according to the results of a pre-test (without GLP) which showed that the solution equilibrium was reached after this time. In this pre-test the same test item concentrations were analytically measured in filtrates after stirring for 3, 24 and 96 hours.
Then, the supersaturated dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) just before the preparation of the test medium.
The undiluted filtrate of the supersaturated dispersion with the maximum concentrafion of dissolved test item was used as the highest concentrated test medium. Additionally, adequate volumes of the filtrate were diluted with test water for the preparation of test media with lower test item concentrations. No additional dilution step was inserted. The test media were prepared just before addition of algae (= start of the test). The actual concentrations of the test item in the test media were analytically determined.
The test concentrations were based on the results of a range-finding test and the results of pre-experiments to the solubility of the test item (without GLP).
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
The test organisms used for the study was Scenedesmus subspicatus CHODAT, strain no.: 86.81 SAG, supplied by the Sammlung von Algenkulturen Göttingen (SAG, Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, 37073 Göttingen, Germany). The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
23°C
pH:
Start of the test (t = 0h): 7.9-8.0
End of the test 8t = 72h): 9.0-9.5
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations: a filtrate of a supersaturated dispersion with a loading rate of 100 mg/L, and dilutions 1:2, 1:4, 1:8 and 1:16
Measured concentrations:
- start of the test (t = 0h): 0.165 mg/L, 0.106 mg/L, 0.0549 mg/L, 0.0283 mg/L and 0.0142 mg/L, respectively
- end of the test (t = 72h): 0.015 mg/L for the undiluted filtrate (9% of the initially measured value), the lower test concentrations were not analyzed since they were belwothe 72-hour NOEC

The losses could be due to photosensitivity of the test item.
Details on test conditions:
TEST SYSTEM
- The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients: NaHCO3: 50.0 mg/L, CaCl2 x 2 H2O: 18.0 mg/L, NH4Cl: 15.0 mg/L, MgSO4 x 7 H2O: 15.0 mg/L, MgCl2 x 6 H2O: 12.0 mg/L, KH2PO4: 1.6 mg/L
Trace elements: Na2EDTA x 2 H2O: 100.0 μg/L, FeCl3 x 6 H2O: 80.0 μg/L, MnCl2 x 4 H2O: 415.0 μg/L, H3BO3: 185.0 μg/L, Na2MoO4 x 2 H2O: 7.0 μg/L, ZnCl2: 3.0 μg/L, CoCl2 x 6 H2: 1.5 μg/L, CuCl2 x 2 H2O: 0.01 μg/L
- The test was started (t = 0h) by inoculation of 10 000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test under the same conditions as in th test.
- The test design included three replicates per test concentration and six replicates of the control.
- Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.
- The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 23°C, and continuously illuminated at the measured light intensity of about 8600 Lux (mean value), range: 7860-9120 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Counting and examination of the algal cells:
Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours exposure and were not replaced. The algal densities in the samples were determined with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.
In addition, after 72 hours exposure, a sample was taken from the control and the undiluted filtrate to examine the shape of the algal cells microscopically.

TEST CONCENTRATIONS
According to the results of a pre-experiment (without GLP) the test item was not soluble at a concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared. Therefore, a filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilutions 1:2, 1:4, 1:8 and 1:16 were tested.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
The mean algal cell densities in the test medium were at all counting dates equal or even higher than those in the control cultures.
Thus, the test item respectively its degradation products had clearly no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours in the undiluted filtrate with the a loading rate of 100 mg/L (mean measured concentration of 90 μg/L). This test concentration was therefore determined as the 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours). The NOEC might
even be higher but concentrations in excess of 90 μg/L could not be tested due to the low water solubility of the test item.

The 72-hour LOEC (lowest concentration tested with toxic effects), respectively the 72-hour EC50 for the mean algal biomass and the mean growth rate were clearly higher than a loading rate of 100 mg/L (mean measured concentration of 90 μg/L). These values could not be quantified, since the test item had no toxic effect on the alga up to the highest test item concentration which could be dissolved in the test water.
In conclusion, the test substance and its degradafion products had no toxic effect on the algae up to its solubility limit in test water.

The microscopic examination of the algal cells after 72 hours test period showed no difference between the algae growing in the undiluted filtrate (loading rate 100 mg/L) and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at up to this test concentration were obviously not affected. In the control the cell density has increased from nominal N = 1E4 cells/mL at the start of the test (0 hours) to N = 49E4 cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled.
No remarkable observations were made concerning the appearance of test media. The test media of all test concentrations remained clear throughout the entire test period.
Validity criteria fulfilled:
yes
Conclusions:
No toxic effects occur within the range of solubility. The test substance is with high probability nat acutely harmful to aquatic algae.
Executive summary:

In this guidleine (OECD 201) study conducted with GLP certification, the 72 hour EC50 of the test material (EC 438-340-0) to algae (Desmodesmus subspicatus) was determined to have no toxic effects within the range of solubility. The test was conducted in freshwater, static, conditions. Test concentrations were prepared with a super-saturated nominal concentration of 100 mg/L, and then diluted 1:2, 1:4, 1:8 and 1:16. The result of this test is not sufficient to trigger classification and labelling under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Description of key information

Study conducted to recognised testing guidelines with GLP.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

No toxic effects occur within the range of solubility. The test substance is with high probability not acutely harmful to aquatic algae.

The study was performed according to OECD guideline (RCC Ltd. 2002j). Due to the low solubility of the test substance in the test medium a saturated solution has been used. The 72-hour NOEC and the 72-hour EC0for the test substance toScenedesmus subspicatuswere determined to be at least at the loading rate of 100 mg/L (mean measured concentration: 90 µg/L). The 72-hour NOEC and the 72-hour EC0might even be higher but loading rates in excess of 100 mg/L were not tested.

The 72-hour EC50and the 72-hour EC100were clearly higher than the loading rate of 100 mg/L. These values could not be quantified since the test item had no toxic effect on the on the algae (mean algal biomass and mean growth rate) up to 100 mg/L.