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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:
2010-01-11 till 2010-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Principles of method if other than guideline:
The purpose of this method was to determine those concentrations which caused a 10 % and a 50 % adverse effect (= EC 10, EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum test concentration of 100 mg/L or at the limit of water solubility, respectively. Effect data are expressed on the basis of yield [y] and growth rate [r].
A range-finding (non-GLP) test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range-finding test: 0.1, 0.32, 1.0 and 2.7 mg/L.
In the main test, the algae were exposed to the test item added to water at a range of concentrations for a period of 72 hours. Defined concentrations of the test item led to a certain inhibition of algal yield and growth rate at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. The 72 hour EC 10 and EC 50 (based on both, yield [y] and growth rate [r]), were calculated by probit analysis.
All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 19.02.2009). For the calculations all algae counts were divided by a factor of 10000.
During the test a temperature range of 21 - 24 °C was maintained in the test vessels.
The pH was measured at the beginning of the test and after 72 hours of exposure.
The maintenance of test item concentrations was proved by analytical measurements. In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using replicate I of the test.
In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling schedule: concentrations of 0.05, 0.1, 0.2, 0.4 and 0.8 mg/L (nominal), control and a concentration of 0.8 mg/L without algae were measured at 48 and 72 hours. The stock solution was measured at 0 hours.
- Sample storage conditions before analysis: 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:
Pre-treatment of the test item
To accelerate the solution procedure, a five fold amount of the water solubility of the test item (4 mg/L) was taken (20.2 mg/L) and added directly to 1 litre of dilution water and treated for 60 sec. at 8000 rpm with an ultra-turrax and afterwards stirred for 24 hours on a magnetic stirrer. Finally undissolved particles of the test item were removed by filtration using a folded filter of pore size 7-12 µm. The pH was measured to be pH 7.9. To produce the different test item concentrations the prepared stock solution was diluted with dilution water to a volume of 100 mL/replicate. For each test item concentration and for the control 3 replicates were prepared.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus (formerly Scendedesmus subspicatus)
- Source (laboratory culture): Strain of the test species obtained from the 'Collection of Algal Cell cultures' of the Plant Physiology at the University of Göttingen (Germany)
- Maintenance and 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 mE-2/s (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.
- Prepearation of pre-cultures: Pre-cultures were set up three days before the start of the test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium. a gowth medium according to the OECD medium of OECD TG 201 was used.
- Test cultures: The algal inocula for a test were taken from an exponentially-growing pre-culture. An amount of 0.806 mL of algal inoculum was mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Any deformed or abnormal cells observed: not indicated
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mh/L CaCO3.
Test temperature:
21-24 °C
pH:
Test start: 7.8
Test end: 7.7 - 7.8
Nominal and measured concentrations:
Nominal concentrations: 0.05, 0.1, 0.2, 0.4 and 0.8 mg/L
Measured concentrations: 0.01, 0.02, 0.038, 0.0835, 0.1597 mg/L (concentrations are given as geometric mean measured values)
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask with cotton stoppers
- Material, size, headspace, fill volume: Test item concentrations were diluted to a volume of 100 mL/replicate. Afterwards 0.806 mL of algal inoculum were added resulting the final cell density.
- Initial cells density: 5000 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
A standard medium according to OECD TG201 was used.

OTHER TEST CONDITIONS
- Temperature was measured and recorded daily in a water filled flask which was inoculated under the same conditions as the test flasks.
- Adjustment of pH: none
- Photoperiod: continuous and uniform illumination in the spectral range of 400 to 700 nm
- Light intensity and quality: ca. 4000 - 8000 lx

EFFECT PARAMETERS MEASURED:
Item-induced inhibition of yield (y) and growth rate (r) of the algal population. Cell concentrations were determined using a Microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask for measurements, which were not replaced.

TEST CONCENTRATIONS
- Range finding study: A non-GLP range-finding study preceded the main test. The following nominal concentrations of the test item were tested: 0.1, 0.32, 1.0 and 2.7 mg/L.
- Spacing factor for test concentrations: 2
- Nominal concentrations: 0.05, 0.1, 0.2, 0.4 and 0.8 mg/L
- Measured concentrations: 0.01, 0.02, 0.038, 0.0835, 0.1597 mg/L (concentrations are given as geometric mean measured values)
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErC10
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEC [r]
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (tα 0.05)
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
0.025 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.0245-0.0257
Duration:
72 h
Dose descriptor:
other: EyC10
Effect conc.:
0.018 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.0176-0.0185
Duration:
72 h
Dose descriptor:
other: NOEC[y]
Effect conc.:
0.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (tα 0.05)
Details on results:
The results are expressed in terms of geometric mean measured concentrations. Effective concentrations were measured as follows:
76.8 - 80.3 % of nominal values at 0 hours
21.5 - 26.8 % of nominal values at 24 hours
12.0 - 14.8 % of nominal values at 48 hours
6.0 - 7.1 % of nominal values at 72 hours
Reported statistics and error estimates:
EC50 and EC10 values for both, yield (y) and growth (r), were calculated by probit analysis. The percentage inhibition of yield and the growth rate were calculated in accordance with the formulas provided in the OECD TG 201. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 19.02.2009). For the calculations all algae counts were divded by a factor of 10000.

While the study report gives a NOEC value it clearly marks it as invalid as it may not be mathematically derived. There is no inhibition at the NOEC concentration but even a slight increase in algal growth rate.
Validity criteria fulfilled:
yes
Remarks:
(- The factor of biomass parameter 121.0 >16; - The mean of the replicate coefficient of variation in the section-by-section growth 18.9 % <35 %; - The coefficient of variation of hte mean specific growth rate replicates in the control 0.7 % <7 %.)
Conclusions:
The toxicity of the substance to algae was measured during a period of 72 hours. The growth rate inhibition resulted in an ErC10 of 0.02 mg/L and an ErC50 of 0.035 mg/L and both values can be used for regulatory purposes. While the study report additionally gives a NOEC[r] of 0.01 mg/L it was clearly stated that the NOEC is not valid as it could not be derived mathematically (statistically not possible).
Executive summary:

The toxicity of the substance to algae was assessed in a 72h-growth inhibition test according to the OECD 201 (2006). Desmodesmus subspicatus was used as test species and the nominal concentrations tested were 0.05, 0.1, 0.2, 0.4 and 0.8 mg/L. The criteria of adverse effects used in the present study were item-induced inhibition of yield and growth rate of the algal population determined by measuring the cell density in a microcell counter. Cell densities were measured at 24h intervals. Since the deviation from the initial concentration was not within the range of +/- 20%, analysis on the results are based on the geometric mean concentration during exposure. The 72h-EC10 for growth was determined to be 0.02 mg/L and the 72h-EC50 for growth was determined to be 0.035 mg/L. The NOEC was found to be invalid.

Description of key information

The toxicity of the substance to algae was measured during a period of 72 hours and the growth inhibition showed an ErC10 of 0.02 mg/L and an ErC50 of 0.035 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:
0.035 mg/L
EC10 or NOEC for freshwater algae:
0.02 mg/L

Additional information

The toxicity of the substance to algae was assessed in a 72h-growth inhibition test according to the OECD 201 (2006). Desmodesmus subspicatus was used as test species and the nominal concentrations tested were 0.05, 0.1, 0.2, 0.4 and 0.8 mg/L. The criteria for adverse effects used in the present study were item-induced inhibition of yield and growth rate of the algal population determined by measuring the cell density in a microcell counter. Cell densities were measured at 24h intervals. Since the deviation from the initial concentration is not within the range of +/- 20%, analysis on the results are based on the geometric mean concentration during exposure. The 72h-EC10 for growth was determined to be 0.02 mg/L and the 72h-EC50 for growth was determined to be 0.035 mg/L. The NOEC was found to be 0.01 mg/L.

The study report also gives a NOEC but clearly states that this value is invalid as it could not be mathematically derived using valid statistical software (ToxRatPro Version 2.10 (released 19.02.2009)). In fact, it was only read from the table which gives concentrations tested (Table 8, p. 27). At the NOEC concentration there was even a slight increase in algal growth instead of an inhibition. The use of ErC10 instead of NOEC would, therefore, be preferred.

The use of EC10 instead of NOEC is also supported by ECHA Guidance Document R.10 (May 2008):

- In general, this Guidance Document always asks to use “EC10 or NOEC” which underlines that, basically, both values may be used for the assessment.

- On page 8 it is highlighted that “The value of the NOEC is limited to being one of the tested concentrations (i.e. if different values were chosen for the tested concentrations, the value of the NOEC would be different).” However, the concentration space is very close in the present study: 0.05, 0.1, 0.2, 0.4 nd 0.8 mg/L and only an EC10 at 0.2 mg/L could be mathematically determined.

- Furthermore, R.10 on page 8 highlights “If the power [of a test] is high, it may occur that biologically unimportant differences are found to be statistically significantly different.” The statistical power of the available GLP and OECD 201 study is high but a NOEC could, nevertheless, even not be calculated.

- EC10 and NOEC both use the same assessment factors to derive the PNECs (e.g. in Table R.10-4).

- Page 11 (Table R.10-1) section “long-term studies” gives: “An EC10 for a long-term test which is obtained using an appropriate statistical method (usually regression analysis) will be used preferentially.”

Furthermore, CLP Regulation (EC) No 1272/2008 (31.12.2008), section 4.1.2.7. Aquatic toxicity, subsection 4.1.2.7.2., gives: “For determining chronic aquatic toxicity for classification purposes data generated according to the standardised test methods referred to in Article 8(3) shall be accepted, as well as results obtained from other validated and internationally accepted test methods. The NOECS or other equivalent L(E)Cx (e.g. EC10) shall be used.” This statement is important and has to be kept in mind as most tables only give NOEC and not additionally ECx to derive the environmental classification of a substance.

Finally, OECD 201 (23 March 2006) “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, which is the base for determining NOEC or ErCx of the present algal study, in its section on statistical procedures, No. 58, gives: “For determining chronic aquatic toxicity for classification purposes data generated according to the standardised test methods referred to in Article 8(3) shall be accepted, as well as results obtained from other validated and internationally accepted test methods. The NOECS or other equivalent L(E)Cx (e.g. EC10) shall be used.”

And Annex 1 of OECD 201 defines LOEC as: “Lowest Observed Effect Concentration (LOEC) is the lowest tested concentration at which the substance is observed to have a statistically significant reducing effect on growth (at p < 0.05) when compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.”

And last but not least, taking into account that technical advancement has led to low variance in control as well as in test concentrations studies need to be critically evaluated if statistically significant deviations are also biologically relevant. Small deviations might result in statistical significance but positive biological significance at low inhibition levels is questionable. USEPA, e.g., gives that NOEC shall be determined at 10-30% inhibition but not at lower inhibitions.

Concluding, for ditolyl ether there is a GLP OECD 201 study on algal growth inhibition for which only an ErC10 can mathematically be determined. The NOEC was given to be invalid in the study. However, ECHA Guidance R.10 gives that ErC10 can be used instead of NOEC (for PNEC derivation). Furthermore, there is an indication that NOEC might even be critical for regulatory purposes from due to conflict between statistical and biological relevance. Therefore, only ErC10 is valid and shall be used for regulatory purposes of ditolyl ether.

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