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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:
16 December 2016 - 5 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No: M1605-05371-01
Storage: At room temperature, ca. 20°C
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicate samples per test concentration)
- Blank control (Bn); containing test medium and algae (six replicate samples)
- Because the test item is a mixture with a limited solubility, only the water-soluble fractions (WSFs) was tested. These WSFs were prepared separately for each test concentration, by adding the respective amounts of test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware. The test item was added by using a Hamilton pipette, considering a density of 1.12 g/ml. These mixtures were moderately stirred for 96 h to maximize dissolution. Subsequently, the WSFs were left standing for about an hour to allow for separation of the test item from the water phase. The aqueous phase was then sampled from the bottom by an outlet, as the test item swims on the top. Of each resulting test solution, 100 ml were added to sterile test flasks and inoculated with an exponentially growing preculture of algae..
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Because the test item is a mixture with a limited solubility, only the water-soluble fractions (WSFs) was tested.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: None – the culture is re-purchased annually
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
Min:22.8; Max: 23.8; Mean: 23.2
pH:
7.1 - 8.1
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
The concentrations of the three main components in the test item were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses confirmed their limited and different solubilities, and showed that they all were decreasing over the whole 72-h test period. While the sum of the three components accounted for 63, 54, 43, 38 and 31% of the respectively 100, 56.2, 31.6, 17.8 and 10.0 mg/l loadings, it dropped down to 27, 18, 11, 8 and 7% after 72 hours of exposure.
Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations of the sum of the three main components over the whole 72 h test period.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm)
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of the three test items was performed. These
measurements showed that the three main test item components were only dissolved to about 50% of the nominal loading, and that the determined concentrations decreased over the 72-h test period.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.
Reference substance (positive control):
not required
Remarks:
Control of sensitivity: None – the culture is re-purchased annually
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.95 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 78% at 100 mg/l and 45% at 56.2 mg/l loading. No significant effects were observed at 31.6, 17.8 and 10.0 mg/l loading.
Based on these effects and the measured concentrations of the test item, the median effect concentration with respect to growth rate (ErC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 20.7 mg/l (95% confidence limits: 19.0–22.5 mg/l).
The low effect concentration with respect to growth rate ErC10 was calculated to be 7.45 mg/l (95% confidence limits: 5.97–8.77 mg/l)..
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 6.95 mg/l.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 98% at 100 mg/l, 86% 56.2 mg/l and 12% at 31.6 mg/l loading. No significant effects were observed at 17.8 and 10.0 mg/l loading.
Based on these effects and the measured concentrations of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 11.3 mg/l (95% confidence limits: 10.5–12.0 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 6.60 mg/l (95% confidence limits: 5.86–7.26 mg/l)..
The no observed effect concentration (NOEyC) with respect to yield was determined to be 3.23 mg/l.
Reported statistics and error estimates:
Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.
Validity criteria fulfilled:
yes
Remarks:
In the control cultures: average specific growth rate of 1.38 (>0.92)/ coefficient of variation of average specific growth rates: 1% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 16.8% (<=35%).
Conclusions:
The acute toxicity of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Desmodesmus subspicatus was estimated to be 20.7 mg/l based on the measured concentrations. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 6.95 mg/l based on the measured concentrations. The results of the test can be considered reliable without restriction.
Executive summary:

The effective concentrations (ErC10, ErC50 and EyC10, EyC50) as well as the no observed effect concentrations (NOErC and NOEyC), of reaction mass of N-butylphthalimide and N-propylphthalimide

and N-sec-butylphthalimide to the green alga Desmodesmus subspicatus, based on the average specific growth rate and the yield (algal biomass production), were investigated over a period of 72 h, following the guideline OECD 201.

The test item is liquid, poorly soluble and consists of a reaction mass of three components, which account for a total purity of 99.8%. Consequently, the test solutions were prepared as 96-h WSFs (water soluble fractions), separately for each test concentration.

The loading rates (test concentrations) were 100, 56.2, 31.6, 17.8 and 10.0 mg/l.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The concentrations of the three main components of the test item in the test media were determined by HPLC analysis at the beginning of the test and after 24, 48 h and 72 h of exposure. These analyses confirmed their limited and different solubilities, and showed that they all were decreasing over the 72-h test period. While the sum of the three components accounted for 63, 54, 43, 38 and 21% of the respectively 100, 56.2, 31.6, 17.8 and 10.0 mg/l loadings, it dropped down to 27, 18, 11, 8 and 7% after 72 hours of exposure. Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations of the sum of the three main components over the whole 72 h test period.

The algal growth inhibition was assessed as two endpoints, growth rate and yield, after 24, 48 and 72 hours of exposure.

The results of the growth rate (r) and yield (y) inhibition of Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to the green alga Desmodesmus subspicatus are summarized in the following table showing EC values and 95% confidence limits (cl) based on the measured concentrations:

 Parameter, expressed as Measured concentration (mg/l)

 ErC10

 ErC50

 NOErC

 Estimate

 7.45

 20.7

 6.95

 Lower 95%-cl

 5.97

 19.0

 

 Upper 95%-cl

8.77

 22.5

 

 Parameter, expressed as Measured concentration (mg/l)

 EyC10

 EyC50

 NOEyC

 Estimate

 6.60

 11.3

 3.23

 Lower 95%-cl

5.86

 10.5

 

 Upper 95%-cl

 7.26

12.0

 

cl: confidence limit

The 72 h ErC50 and EyC50 values of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide on the green alga Desmodesmus subspicatus were 20.7 mg/l and 11.3 mg/l, respectively. These values are based on measured concentrations.

All validity criteria were fulfilled

Description of key information

The acute toxicity of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide to Desmodesmus subspicatus was estimated to be 20.7 mg/l based on the measured concentrations. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 6.95 mg/l based on the measured concentrations. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

Additional information