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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:
25 September 2002 to 08 November 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the start of the growth inhibition test, duplicate samples were taken from all media containing all nominal test material concentrations tested together with a control. At the end of the test, duplicate samples were taken from the media containing the same test material concentrations with algae. The sample volume was 100 mL except for the highest test material concentration where the sample volume was 10 mL. One of the duplicate sample series at the start and at the end of the test was analysed.
Vehicle:
no
Details on test solutions:
40.8 mg test material was dissolved in 250 mL algal medium. Dilutions were then prepared in medium so as to yield the final test material concentration series.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
The fresh-water green alga Scenedesmus subspicatus (SAG 86.81) was used as the test organism. The culture was supplied by the Collection of Algal Cultures, Institute for Plant Physiology, University of Gottingen, Nikolausberger Weg 18, D-37073 Gottingen, Germany.

A pre-culture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, using the medium described in 2.4. The initial inoculum cell density was 0.3 x 10E4 cells/ml to obtain the appropriate biomass level in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable.
Hardness:
Hardness, mg equivalent CaCO3/L = 24.2
Test temperature:
23.7-23.9 °C
pH:
At the beginning of the test - 7.9-8.0
At the end of the test - 8.0-8.1
Nominal and measured concentrations:
At the beginning of the test - 0, 0.004, 0.020, 0.053, 0.170, 0.50, 2.1 mg/L (measured)

0, 0.01, 0.03, 0.10, 0.32, 1.0, 3.2 mg/L (nominal)
Details on test conditions:
The medium was prepared from concentrated stock solutions in ultra pure water. It was sterilised by micropore filtration and contained 150 mg/L NaHCO3 (not 50 mg/L as specified in the OECD Guideline, this in order to improve the buffer capacity of the medium). Furthermore, the medium contained Fe-citrate because the growth of the algae can become erratic in the absence of complexed iron. The following solutions of test material were made:

-For the range-finding test, an amount of 100.6 mg test material was dissolved in 1000 mL algal medium. Dilutions were then prepared in medium so as to yield the final test material concentration series. Nominal inoculcum cell density was 0.8 x 10E4 cells/mL.
*The test was carried out in duplicate with four controls containing algae only, and a single background series containing test substance without algae

-For the growth inhibition test, an amount of 40.8 mg test material was dissolved in 250 mL algal medium. Dilutions were then prepared in medium so as to yield the final test material concentration series. Nominal inoculum cell density of 0.3 x 10E4 cells/mL.
* The test was carried out in triplicate with six controls containing algae only and a single background concentration series containing test material without algae

Light intensity = 63-72 umol/s/m^-2

Algal cell density and biovolume were determined. Both techniques of measurement of algal biomass are considered applicable in the OECD 201 test guideline. Algal cell density refers to the number of algal cells counted per unit volume of test media (mL) and biovolume refers to the total volume of algal cells in that given unit volume (mL). Biovolume was used to calculate effects concentrations/ endpoints due to a slight difference in cell size in some test concentrations.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (geom. mean)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Basis for effect:
growth rate
Remarks on result:
other: 0.25-0.40 (95% confidence limits)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.107 mg/L
Nominal / measured:
meas. (geom. mean)
Basis for effect:
biomass
Remarks on result:
other: 0.04-0.13 (95% confidence limits)
Details on results:
The range-finding test revealed that inhibiting effects could be expected at test substance concentrations of >0.01 mg/L.
Reported statistics and error estimates:
EC values with respect to growth rate during and exponential growth were calculated by means of a parametric model developed by Kooijman et al, assuming a constant error per measurement. EC values with respct to the area under the growth curve were calculated by the method given in the OECD guideline. The values were calculated by linear interpolation of a plot of the percentage reduction in growth against the log concentration of the test material.

The NOEC was determined as the highest concentration at which no statistically significant inhibition was observed. Model calculations carried out using the DEBtox software package according to Dynamic Energy Budgets Theory developed by Kooijman and Bedaux. Model parameters for population growth and their asymptotic standard deviation and correlation coefficients were estimated. The NEC was calculated from the Profile Ln Likelihood Function.

Table 1. Results of the analysis of the concentration of MBTC in the algal test media at the start and end of the test:

Nominal Concentration (mg/L) Measured concentration of the Test Material
0 h (mg/L) % of nominal 68 h (mg/L) % of nominal Geometric mean (mg/L) Geometric mean (% of nominal)
0.00 <0.002 - <0.002 - - -
0.01 0.004 40.0 0.003 30.0 0.004 41
0.03 0.020 62.5 0.014 43.8 0.012 41
0.10 0.053 53.0 0.041 41.0 0.040 42
0.32 0.170 53.1 0.088 27.5 0.130 42
1.0 0.500 50.0 0.200 20.0 0.430 43
3.2 2.100 65.6 1.360 42.5 1.380 43

Table 2. Mean particle size (µm3, corrected for background) in the growth inhibition test.

Time (h) Nominal concentration of the Test Material (mg/L)
0 0 0.01 0.03 0.10 0.32 1.00 3.20
0 112 112 112 112 112 112 112 112
23 120 122 118 123 107 88 92 89
44 105 106 113 124 125 110 80 77
68 78 82 99 132 148 117 57 69
Validity criteria fulfilled:
yes
Conclusions:
Results of the 72-h growth inhibition test of the test material: NOEC = 0.012 mg/L, ErC50 = 0.31 (0.25 -0.40) mg/L and EbC50 = 0.107 (0.04 -0.13) mg/L.
Executive summary:

The toxicity of the test material to the fresh water green alga Scenedesmus subspicatus was determined in a 72 -h growth inhibition test according to OCED 201 and EU Guideline C3. The test fulfilled the validity criteria listed in the guideline. Growth was sufficient and there was a minimal increase of the test media pH values.

The nominal concentrations of the test material were 0, 0.01, 0.03, 0.10, 0.32, 1.0 and 3.2 mg/L and were added to the algal growth medium. The measured concentrations of the test material at the start of the test were 0, 0.004, 0.020, 0.053, 0.170, 0.05 and 2.1 mg/L and these values were 40, 62.5, 53, 53.1, 50 and 65.6 % respectively of the nominal concentrations. The measured concentrations of the test material at the end of the test were 30, 43.8, 41, 27.5, 20 and 42.5 % respectively of the nominal concentrations. The geometric means of the measured concentrations of the test material at the start and end of the test were used for the calculation of the effect values.

Results of the 72-h growth inhibition test of the test material: NOEC = 0.012 mg/L, ErC50 = 0.31 (0.25 -0.40) mg/L and EbC50 = 0.107 (0.04 -0.13) mg/L.

The statistical assessment of these data support the conclusion that there was a significant effect only on the growth rate of S. subspicatus in a 72-h growth inhibition test and it is concluded that the ErC50 value of 0.31 mg/L based on measured concentration of the test material represents the best estimate of the mean effect concentration of the test material on algal growth. The NOEC of the test material was the measured concentration of 0.012 mg/L.

Description of key information

Oldersma (2003)

NOEC = 0.012 mg/L, ErC50= 0.31 (0.25 -0.40) mg/L and EbC50= 0.107 (0.04 -0.13) mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.31 mg/L
EC10 or NOEC for freshwater algae:
0.012 mg/L

Additional information

Oldersma et al, 2003 was selected as the key study for this endpoint and for classification and labelling.

The toxicity of the test material to the fresh water green alga Scenedesmus subspicatus was determined in a 72 -h growth inhibition test according to OCED 201 and EU Guideline C3. The test fulfilled the validity criteria listed in the guideline. Growth was sufficient and there was a minimal increase of the test media pH values.

The nominal concentrations of the test material were 0, 0.01, 0.03, 0.10, 0.32, 1.0 and 3.2 mg/L and were added to the algal growth medium. The measured concentrations of the test material at the start of the test were 0, 0.004, 0.020, 0.053, 0.170, 0.05 and 2.1 mg/L and these values were 40, 62.5, 53, 53.1, 50 and 65.6 % respectively of the nominal concentrations. The measured concentrations of the test material at the end of the test were 30, 43.8, 41, 27.5, 20 and 42.5 % respectively of the nominal concentrations. The geometric means of the measured concentrations of the test material at the start and end of the test were used for the calculation of the effect values.

Results of the 72-h growth inhibition test of the test material: NOEC = 0.012 mg/L, ErC50= 0.31 (0.25 -0.40) mg/L and EbC50= 0.107 (0.04 -0.13) mg/L.

The statistical assessment of these data support the conclusion that there was a significant effect only on the growth rate of S. subspicatus in a 72-h growth inhibition test and it is concluded that the ErC50value of 0.31 mg/L based on measured concentration of the test material represents the best estimate of the mean effect concentration of the test material on algal growth. The NOEC of the test material was the measured concentration of 0.012 mg/L.