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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 20 to November 10, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Complete read across justification is attached in section 13. Source study has reliability 1.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For the analysis of the actual test item concentrations, the samples were taken just before test start:
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
and after 72 hours (stability samples):
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)

For the 72-hour stability samples, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
All the samples as listed below were analyzed immediately after sampling since a reliable examination of the storage stability was not possible due to the very low solubility of the test item in test water.
The concentrations of the test item were analyzed in the test medium samples from both sampling times (0 and 72 hours). From the control samples, only one of the duplicate samples was analyzed from each of both sampling times.
The analytical results were corrected by the recoveries obtained for the spiked samples. The analytical procedure and results are described in the attached analytical report.
Vehicle:
no
Details on test solutions:
According to the results of a pre-experiment (without GLP), the test item is nearly insoluble in test water.
Therefore, a filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 were tested. Additionally, a control was tested in parallel (test water without test item).
A supersaturated dispersion with a concentration of nominal 100 mg/l (loading rate) was prepared by weighing 450 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 stirring for 7 days at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
The long stirring period of the dispersion of 7 days was chosen according to the results of a pre-test (without GLP) which showed that the test item is hardly soluble in test water.
The supersaturated dispersion of the test item was filtered through a glass microfibre filter (Whatman GF/C, maximum pore size approximately 1.2 µm) after the stirring period of 7 days just before the preparation of the test media. A filter with a pore size of approximately 1.2 µm was chosen since the test item concentrations in a filtrate filtered over a membrane filter with a smaller pore size of 0.45 µm were below the limit of quantification of the analytical method (pre-experiment without GLP). The undiluted filtrate of the supersaturated stock dispersion with the maximum concentration of dissolved and very finely dispersed 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.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: no. 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, D-37073 Göttingen
- Method of cultivation: according to test guidelines

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as in the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/l = 24 mg/l CaCO3
Test temperature:
22 °C
Nominal and measured concentrations:
The measured test item concentrations in the test media samples (dilutions 1:22, 1:10, 1:4.6, 1:2.2, and the undiluted filtrate) amounted to 0.0023, 0.0049, 0.0077, 0.016, and 0.027 mg/l at the start of the test.
In these test media, incubated under the test conditions during the test period (but without algae), the measured concentrations decreased to 0.0009, 0.0018, 0.0026, 0.006, and 0.010 mg/l after 72 hours (34-38 % of the initially measured concentrations). This decrease of the test item concentrations might have been due to a loss by adsorption onto the glass surfaces or photolytical degradation of the test item.
Details on test conditions:
TEST SYSTEM
- Type: flasks covered with glass dishes
- Initial cells density: 10000 algal cells per ml test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM: same as test medium

TEST MEDIUM / WATER PARAMETERS
Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/l
CaCI2 × 2H2O 18 mg/l
NH4CI 15 mg/l
MgSO4 × 7H2O 15 mg/l
MgCI2 × 6H2O 12 mg/l
KH2PO4 1.6 mg/l
Trace elements:
Na2EDTA × 2H2O 100 µg/l
FeCI3 × 6H2O 80 µg/l
MnCI2 × 4H2O 415 µg/l
H3BO3 185 µg/l
Na2MoO4 × 2H2O 7 µg/l
ZnCI2 3 µg/l
CoCI2 × 6H2O 1.5 µg/l
CuCI2 × 2H2O 0.01 µg/l

OTHER TEST CONDITIONS
- Photoperiod: continous illumination
- Light intensity and quality: ca. 8600 Lux (mean value), range 8000 to 9200 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media). Illumination was achieved by fluorescent tubes (philips TLD 36W/840), installed above the flasks.

EFFECT PARAMETERS MEASURED (24, 48 and 72 h of exposure): algal growth inhibition evaluated in terms of growth rate (µ) and biomass (b)
- Determination of cell concentrations: electronic particle counter
- Other: after 72 h, shape of algal cells from control and undiluted filtrate samples was microscopicallt examined

TEST CONCENTRATIONS: based on results of a range-finding and of a first main test and on the results of a pre-experiment to the solubility of the test item (without GLP).
However, loading rates in excess of nominal 100 mg/l or test item concentrations above the maximum concentration which could be dissolved or very finely dispersed in test water were not tested according to the guidelines.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Test item had no statistically significant inhibitory effect on the growth of Scenedesmus subspicatus. The only exception was a statistically significant inhibition of the growth rate after the exposure period of 72 hours at the highest test concentration of 0.018 mg/l in the undiluted filtrate. However, this inhibition of 2.6 % was very small and the inhibition of the mean biomass after the same exposure period was statistically not significantly smaller than the control value. Therefore, this statistically significant result was caused by the very low variability of the biological results between the replicates. An inhibition of 2.6 % can be considered to be of no biological relevance and within the normal range of variability of the results of an algal growth inhibition test.
Thus, the undiluted filtrate with a mean measured test item concentration of 0.018 mg/l (loading rate of 100 mg/l) was therefore determined as the 72-hour NOEC. The NOEC might even be higher but concentrations of the test item above the maximum concentration which could be dissolved or very finely dispersed in the test water were not tested, according to the guidelines.

The 72-hour LOEC and the 72-hour EC50 for the mean algal biomass and the mean growth rate were clearly higher than the solubility limit of test item in the test water. These values could not be quantified, since the test item had no toxic effect up to the highest concentration, which could be dissolved or very finely dispersed in the 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 and the algal cells in the control. The shape and size of the algal cells growing up to this test concentration were
obviously not affected.
In the control, the cell density has increased from nominal N = 1 × 10^4 cells/ml at the start of the test (0 hours) to N = 106 × 10^4 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 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.

At the start of the test, the pH values of control and test media were in the range of 7.9 - 8.0.
At the end of the test, pH values between 8.9 and 9.2 were measured. This increase of the pH during the test was obviously caused by the CO2-consumption of the algae due to their rapid growth (although the test media have been intensively stirred).

Biomass (AUC = area under curve) and percentage inhibition of biomass (IAUC) during test period

dilution mean measured conc. mg/l 0-24 h 0-48 h 0-72 h
AUC IAUC AUC IAUC AUC IAUC
control - 55 0.0 398 0.0 1943 0.0
1:22 0.0016 47 14.2 415 -4.1 2114 -8.8
1:10 0.034 47 13.5 408 -2.3 2076 -6.9
1:4.6 0.0052 47 13.5 427 -7.2 2195 -13.0
1:2.2 0.011 59 -6.9 420 -5.5 2019 -4.0
undiluted filtrate 0.018 51 6.6 446 -12.1 1901 2.1

% inhibition: increase in growth relative to control

Growth rate µ and percentage of inhibition (Iµ) during test period

dilution mean measured conc. mg/l 0-24 h 0-48 h 0-72 h
µ µ µ
control - 1.71 0.0 1.61 0.0 1.55 0.0
1:22 0.0016 1.59 6.9 1.66 -3.2 1.58 -2.0
1:10 0.034 1.60 6.4 1.65 -2.5 1.58 -1.7
1:4.6 0.0052 1.60 6.4 1.68 -4.3 1.60 -2.9
1:2.2 0.011 1.77 -3.6 1.63 -1.5 1.56 -0.7
undiluted filtrate 0.018 1.66 2.8 1.69 -5.2 1.51 2.6

% inhibition: increase in growth relative to control

Analytical results

dilution day mg/l average corrected by mean recovery of spiked samples
1:22 0 0.00127 0.0023
0.00103
72 0.00067 0.00087
0.00059
mean 0.00089 0.0016
1:10 0 0.00268 0.0049
0.00221
72 0.00139 0.0018
0.00127
mean 0.0019 0.0034
1:4.6 0 0.00449 0.0077
0.00326
72 0.00238 0.0026
0.00138
mean 0.00089 0.0052
1:2.2 0 0.00888 0.0155
0.00663
72 0.00572 0.0061
0.00309
mean 0.0061 0.0108

undiluted filtrate

(0.018 mg/l)

0 0.0128 0.0269
0.0141
72 0.00620 0.0096
0.00768
mean 0.0102 0.0182
Validity criteria fulfilled:
yes
Conclusions:
NOEC (72h) = 0.018 mg/l, i.e. the highest test item concetration, which can be dissolved or very finely dispersed in test water.
Executive summary:

Method

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU method C.3 (1992) and the OECD guideline 201 (1984). Due to the very low water solubility, a supersaturated stock dispersion of the test item with a loading rate of nominal 100 mg/l was continuously stirred at room temperature in the dark over 7 days. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved and very finely dispersed test item and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 were used as test media. Additionally, a control was tested in parallel.

Results

The measured test item concentrations in the test media samples (dilutions 1:22, 1:10, 1:4.6, 1:2.2, and the undiluted filtrate) amounted to 0.0023, 0.0049, 0.0077, 0.016, and 0.027 mg/l at the start of the test. In these test media, incubated under the test conditions during the test period (but without algae), the measured concentrations decreased to 34-38 % of the initially measured concentrations. This decrease of the test item concentrations might have been due to a loss by adsorption onto the glass surfaces or photolytical degradation of the test item. Therefore, all biological results are related to the mean measured test item concentrations (calculated as the average over all measurements per test concentration): 0.0016 mg/l (dilution 1:22), 0.0034 mg/l (dilution 1:10), 0.0052 mg/l (dilution 1:4.6), 0.011 mg/l (dilution 1:2.2), and 0.018 mg/l (undiluted filtrate).

Test item had no inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus during the exposure period of 72 hours up to and including the undiluted filtrate with the mean measured test item concentration of 0.018 mg/l (loading rate of 100 mg/l).

Thus, this test concentration was determined as 72 -hour NOEC. The NOEC might even be higher but concentrations of test item above the maximum concentration which could be dissolved or very finely dispersed in test water were not tested, according to the guidelines. The 72-hour LOEC and the 72-hour EC50 for the algal biomass b and the growth rate µ were clearly higher than the solubility limit of test item in test water. These parameters could not be quantified due to the absence of a toxic effect up to the highest test concentration which could be dissolved or very finely dispersed in the test water.

Description of key information

Highest concentration, which could be dissolved or very finely dispersed: 0.018 mg/l

NOEC (72h) = 0.018 mg/l

LOEC, EC50 (72h) > 0.018 mg/l

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.18 mg/L

Additional information