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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:
2013-04-09 to 2013-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 2006, corrected 2011
Deviations:
yes
Remarks:
The pH in the control increased more during the test period than allowed; temperature during the test was 1 °C higher than the upper limit (24°C) according to OECD 201; deviations do not invalidate the test based on logarithmic growth of control repl.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Preparation: filtration over nylon syringe filters was possible. The samples at the end of the test were filtrated before measurement to remove the alga cells. Samples were diluted before measurement using algal medium.
- Concentrations: all treatments at t=0 h and t=72h
Vehicle:
no
Details on test solutions:
A stock solution containing 100 mg/L test item in nutrient medium (demineralized water enriched with minerals but without algae) was prepared. Because of the volatility of the test item, the nominal load was directly added into the vessel with nutrient medium with a pipette. The lower treatments were prepared by serial dilution of the stock solution in nutrient medium.
To minimise potential evaporation of test item, the test was performed in a closed system. For each treatment, 350 mL of the respective test solution was mixed with the necessary amount of algal pre-culture (1.59 mL for the treatments, 1.81 mL for the blank control which consisted of a higher volume) to achieve a cell concentration of approx. 9000 ceIIs/mL. In this mixture, the pH-value was measured.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: CHODAT
- Source (laboratory, culture collection): Obtained from MBM Sciencebridge GmbH in 09/2011
- Age of inoculum (at test initiation): 72 h
- Method of cultivation: stock cultures kept on solid agar at 8 °C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 - 25 °C
pH:
7.6 - 9.9
For details, see resluts table under "Any other information on results incl. tables".
Nominal and measured concentrations:
Nominal: 0 (control), 4.6, 10, 22, 46, 100 mg/L
Measured geometric mean: 0 (
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: closed glass flasks (total volume: 65 mL)
- Initial cells density: 8824 cells/mL
- Control end cells density: 404139 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium), but to avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL sodium hydrogen carbonate solution (50 g/L) was added to 900 mL nutrient medium (ready to use). Then, pH was adjusted to 7.0 (1 0.2) and the solution was filled up to 1000 mL.

PERFORMANCE OF THE STUDY:
To minimise potential evaporation of test item, the test was performed in a closed system. For each treatment, 350 mL of the respective test solution was mixed with the necessary amount of algal pre-culture (1.59 mL for the treatments, 1.81 mL for the blank control which consisted of a higher volume) to achieve a cell concentration of approx. 9000 cells/mL. In this mixture, the pH-value was measured.
For the control, 400 mL nutrient medium was used instead of test item solution.
The test vessels were completely filled with the respective test solution and incubated closed for 72 hours, shaken on an orbital shaker.
Before the start of incubation and every 24 hours, the cell number was calculated based on the determination of the absorption at 440 nm.

After the test, the pH value in treatments and control was measured again, and the treatments were examined microscopicaly in order to assess the appearance of the alga and detect abnormalities (e.g. caused by the exposure to the test item).

The content of the test item in the test vessels was measured at the start and at the end of the test.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: 5800 lux,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: photometric measurement of optical density at 440 nm every 24 h
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 10-27 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: <6.0 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 2.7-8.3 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: extrapolated; <2.8 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: AUC
Remarks on result:
other: 95% CI: 3.7-13 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: AUC
Remarks on result:
other: 95% CI: extrapolated; <3.9 mg/L
Details on results:
Graphics (concentration - response) are given in the report for inhibition of the growth rate, AUC and yield.
Microscopic observations: at 4.6 and 10 mg/L, cells appeard normal (normal growth). At 22 mg/L, nearly no cells could be observed, while at the higher concentrations no cells were visible at all.

Analytical results of test item concentration monitoring:
Details are given in the table IUCLID section "Any other information on results incl. tables".
The measured concentrations showed good correlation with the nominal concentrations at the beginning of the test. But at the end of the test, the correlation with the nominal concentration and the recovery were only mediocre. In the lowest concentrated treatment (4.6 mgr/L nominal), no test item could be detected after 72 hours. That means that the test item was not stable under test conditions and it might be possible that the measured test item concentration was affected by the preseace of the alga cells (adsorption and/or ingestion) or that abiotic processes took place. Therefore, the determination of the results was based on the geometric
mean of the measured concentrations for treatments 10 - 100 mg/L and for treatment 4.6 mg/L on the half of the measured start concentration.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h ErC50: 0.73 mg/L (range: 0.60 - 1.03 mg/L)
Reported statistics and error estimates:
As no adequate increase of inhibition was observed at the highest treatment (100 mg/L nominal concentration) in all three endpoints (grwoth rate, AUC, yield), this treatment was not used for evaluation of the biological results.
Effect concentration were determined by linear regression on a probability-logarithmic scale: Prob (y)= a+b*log(x), x: concentr. [mg/L]; y: inhib. [%].
Results per endoint:
Growth rate inhibition: Sum of squared residuals: 2.51484; Pearson R = 0.85606; R squared = 0.70316
Inhibition AUC: Sum of squared residuals: 3.47234; Pearson R = 0.82266; R squared = 0.64066
Yield inhibition: Sum of squared residuals: 2.58401; Pearson R = 0.87472; R squared = 0.73905

Statistical testing (growth rate; yield; AUC):
For the treatments with the nominal test item concentrations 4.6 and 10 mg/L (2.26 mg/L and 8.5 mg/L measured concentrations), it was tested whether the differences between treatment and control were significant.
Variance homogeneity was tested using the F-test (95% significance level): F= s1^2/s2^2, s1 being the greater variance and s2 the smaller one. Result: variance homogeneity was given for all three endpoints.
t-Testing:
With the t-Test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with grade of freedom: n1 + n2 - 2, level of significance 95 %).


Analytically verified concentrations

Nominal concentration [mg/L]

Measured concentration [mg/L]

after 0h

Recovery [%]

after 0h based on nominal value

Measured concentration [mg/L]

after 72h

Recovery [%]

after 72h based on nominal value

Geometric mean of measured concentration [mg/L]

0 (control)

0.41

-

<LOQ

-

-

4.6

4.52

98

<LOQ

-

2.26

10

9.08

91

7.88

79

8.50

22

20.93

95

15.51

71

18.0

46

40.82

89

25.87

56

32.5

100

93.44

93

64.32

64

77.5

Inhibition results after 72h

Nominal concentration [mg/L]

Measured concentration, geometric mean [mg/L]

% Inhibiton

Growth rate

Area under the curve AUC

Yield

0 (control)

0

-

-

-

4.6

2.26

6.89

18.8

22.5

10

8.50

15.7

30.0

45.6

22

18.0

38.3

65.5

78.7

46

32.5

86.6

96.3

98.5

100

77.5

89.2

97.7

98.8

Microscopical observations

Nominal concentration [mg/L]

Observation

0 (control)

Normal growth

4.6

Normal growth

10

Normal growth

22

Nearly no cells

46

No cells

100

No cells

pH values

Nominal concentration [mg/L]

0 h

72 h

0 (control)

8.1

9.9

4.6

7.6

9.5

10

7.8

9.5

22

8.2

8.9

46

8.9

8.9

100

9.5

9.5

Validity criteria

Increase factor: The cell concentration in the control should increase by a factor of at least 16 within 72h. The observed value was 46 in this test.

Daily growth rates: Mean coefficient of variation of daily growth rates should be 35% at the most. Coefficient of variation of average growth rate during the whole test period should be 7% at the most. The first value was 19%, the second one 5% in this test.

pH increase: The pH should not change by more than 1.5 units. The change was 1.8 units caused by CO2 consumption in the closed system. As normal growth was observed in the controls and the pH increase in a closed system is a phenomenon observed in studies before, this was stated as uncritical for the outcome of the study.

Validity criteria fulfilled:
yes
Conclusions:
Toxcity of n-butylamine towards freshwater green algae (OECD 201; GLP):
EC50 (growth rate; 72 h; geom. mean measured): 17 mg/L
EC10 (growth rate; 72 h; geom. mean measured): 3.7 mg/L
NOEC (growth rate, yield, AUC; geom. mean measured): 2.3 mg/L
Executive summary:

The submission substance n-butylamine was tested for algal toxicity in a reliable study performed compliant with GLP according to OECD 201. The study was performed using five concentrations ranging from 4.6 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the absorption and the cell density of the cultures determined by microscope counts. Growth rate, area under the growth curve (AUC, integral of the biomass) and the yield were determined from the cell densities at the respective observation times.

Analytical monitoring:

At the start and at the end of the test, the concentration of the test item in the test solutions was determined using HPLC-MS. The measured concentrations at the beginning of the test lay in a range of 89 % to 98 % of the nominal concentration. At the end of the test (after 72 hr), in the lowest concentrated treatment (4.6 mg/L nominal), no test item was detectable. In the other treatments, the measured values lay between 56 % and 79 % (10 mg/L nominal) of the nominal concentrations. As such, it may be considered that the measured test item concentration was affected by the presence of the alga cells (adsorption and/or ingestion). However, recovery did not negatively correlate with algal cell density. Abiotic processes other than adsorption onto algal cell biomass are unlikely because of a very low adsorption potential (experimentally determined Koc from batch-equilibrium test: ca. 49 L/kg). Volatilisation can safely be excluded because the compound is fully ionized at environmental pH based on a pKa of 10.8, and further, closed vessels were used in the test. Finally, biodegradation cannot fully be excluded as the compound was determined to be readily biodegradable. Concluding, because test item stability could not be analytically confirmed to be within +/- 20% of nominal, results are based on the geometric mean measured concentrations for treatments 10 - 100 mg/L and for treatment 4.6 mg/L on the half of the measured concentration determined at the start of the test.

The EC50s of potassium dichromate were tested in a separate reference test (GLP study no. 201301R301). The values lay within the normal range of the laboratory.

The following results were determined in this study:

 

EC50 (growth rate; 72 h; geom. mean measured): 17 mg/L

EC10 (growth rate; 72 h; geom. mean measured): 3.7 mg/L

NOEC (growth rate, yield, AUC; geom. mean measured): 2.3 mg/L

 

EC50 (yield; 72 h; geom. mean measured): 5.6 mg/L

EC10 (yield; 72 h; geom. mean measured): 1.4 mg/L (extrapolated)

 

EC50 (AUC; 72 h; geom. mean measured): 8.0 mg/L

EC10 (AUC; 72 h; geom. mean measured): 1.9 mg/L (extrapolated)

Description of key information

Toxcity of n-butylamine towards freshwater green algae (OECD 201; GLP):

EC50 (growth rate; 72 h; geom. mean measured): 17 mg/L

EC10 (growth rate; 72 h; geom. mean measured): 3.7 mg/L

NOEC (growth rate, yield, AUC; geom. mean measured): 2.3 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
17 mg/L
EC10 or NOEC for freshwater algae:
3.7 mg/L

Additional information

The submission substance n-butylamine was tested for algal toxicity in a reliable study performed compliant with GLP according to OECD 201 (Muckle, 2013). The study was performed using five concentrations ranging from 4.6 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the absorption and the cell density of the cultures determined by microscope counts. Growth rate, area under the growth curve (AUC, integral of the biomass) and the yield were determined from the cell densities at the respective observation times.

Analytical monitoring:

At the start and at the end of the test, the concentration of the test item in the test solutions was determined using HPLC-MS. The measured concentrations at the beginning of the test lay in a range of 89 % to 98 % of the nominal concentration. At the end of the test (after 72 hr), in the lowest concentrated treatment (4.6 mg/L nominal), no test item was detectable. In the other treatments, the measured values lay between 56 % and 79 % (10 mg/L nominal) of the nominal concentrations. As such, it may be considered that the measured test item concentration was affected by the presence of the alga cells (adsorption and/or ingestion). However, recovery did not negatively correlate with algal cell density. Abiotic processes other than adsorption onto algal cell biomass are unlikely because of a very low adsorption potential (experimentally determined Koc from batch-equilibrium test: ca. 49 L/kg). Volatilisation can safely be excluded because the compound is fully ionized at environmental pH based on a pKa of 10.8, and further, closed vessels were used in the test. Finally, biodegradation cannot fully be excluded as the compound was determined to be readily biodegradable. Concluding, because test item stability could not be analytically confirmed to be within +/- 20% of nominal, results are based on the geometric mean measured concentrations for treatments 10 - 100 mg/L and for treatment 4.6 mg/L on the half of the measured concentration determined at the start of the test.

The EC50s of potassium dichromate were tested in a separate reference test (GLP study no. 201301R301). The values lay within the normal range of the laboratory.

The following results were determined in this study:

 

EC50 (growth rate; 72 h; geom. mean measured): 17 mg/L

EC10 (growth rate; 72 h; geom. mean measured): 3.7 mg/L

NOEC (growth rate, yield, AUC; geom. mean measured): 2.3 mg/L

 

EC50 (yield; 72 h; geom. mean measured): 5.6 mg/L

EC10 (yield; 72 h; geom. mean measured): 1.4 mg/L (extrapolated)

 

EC50 (AUC; 72 h; geom. mean measured): 8.0 mg/L

EC10 (AUC; 72 h; geom. mean measured): 1.9 mg/L (extrapolated)