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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:
18 April 2018 to 02 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Storage conditions at the Test Facility: At 20 ± 5 °C under Nitrogen, in the dark
Analytical monitoring:
yes
Details on sampling:
CONCENTRATIONS
Quantification: Samples were quantified by measuring the peak area with reference to the calibration curve. The latter was obtained by correlation of peak area of the standard solutions to their corresponding concentration. The correlation was performed using a linear regression function given by equation (1):
y = a * x + b (1)
where
y: peak area
x: concentration of analyte
a: slope
b: y-axis intercept

Calculated Concentration: The concentration of the analyte in the treatment samples
and in the control sample was calculated by equation (2):
c = x * d (2)
where
c: concentration of analyte in original sample
x: concentration of analyte in the analysed sample
(calculated in equation (1))
d: dilution factor

SAMPLING METHOD
Sampling: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the controls were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the controls (containing algae) were taken at the end of the test (after the 96 hours test period) by pooling the contents of the test beakers of each treatment. The samples remained undiluted until sample preparation which was performed stand-by directly after sampling.

SAMPLE STORAGE CONDITIONS BEFORE ANALYSIS
All samples were analysed stand-by immediately after sampling via Headspace-extraction followed by GC-MS analysis. Therefore, sample storage was not feasible.
Analyses: The concentrations of the test item were analysed in all of the duplicate test media samples from all test concentrations and the control from both sampling times (0 and 96 hours). Since stand-by analysis was performed, test start (analysis 1) and test end samples (analysis 2) were analysed intwo separate analyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 10 mg test item/L was prepared by dissolving 13.7 μL test item into 1000 mL test water by intense stirring for 15 minutes. The pH was adjusted with 1 M HCl from 8.6 to pH 7.9 Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
Control: In the control, test water was used without addition of the test item.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Water: The appearance of the test item in test water was determined daily in all test item concentrations
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV), S
- Strain: train No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).
- Source (laboratory, culture collection): The algae were supplied by the " Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation):
- Method of cultivation: The algae were cultivated in the test laboratory under standardised conditions according to the test guidelines.
- Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

ACCLIMATION
- Acclimation period: Cesll to start the test were taken from an exponentially growing pre-culture, which was set up 3 days prior tot the test start under the same conditions as in the test.
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
0.24mmol/L (=24 mg/L) as CaCO3
Test temperature:
23 to 24°C
pH:
7.9 to 8.1 at test start and 8.5 to 11.2 at test end
Conductivity:
< 5 µScm^-1
Nominal and measured concentrations:
The concentrations tested were 100, 10, 1.0, 0.1 mg test item/L.
Test Concentrations (Definitive Test): 0.1, 0.3, 1.0, 3.2, and 10 mg test item/L corresponding to the following geometric mean concentrations: 0.0667, 0.180, 0.693, 2.79 and 8.44 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible miniumum of some mL).Volumes of approximately 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass sotppers and incubated in a water bath. The flasks were placed in a random order and were repostitioned each dat to minimize differences in test conditions.
- Type (delete if not applicable): Closed
- Initial cells density: 5000 algal cells per mL
- Control end cells density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48, 72 and 96 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test
- No. of organisms per vessel: ~ 250'000 algae per elenmeyer flasks
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): one replicate
- No. of vessels per vehicle control (replicates): one replicate

GROWTH MEDIUM
- Standard medium used: yes Reconstituted Water (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
Macro-nutrients: NaHCO3 50.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace Elements: Na2EDTA × 2 H2O 100.0 μg/L
FeCl3 × 6 H2O 64.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L
- Alkalinity: pH 8.0
- Ca/mg ratio: 0.24 mmol/L (=24 mg/L) as CaCO3
-
OTHER TEST CONDITIONS
- Adjustment of pH:the pH was adjusted with 1 M HCl form 8.3 to pH 8.0
- Light Regime: Continuous illumination
- Lighe Intensity:The light intensity was measured once during the test at 6 positions distributed over the experimental area tat the surface of the test media. Mean light intensity: 7450 lux (range: 7240 to 7650 lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement.

TEST CONCENTRATIONS
- Range finding study: Range-Finding Test: For the determination of the test concentrations a range-finding test was performed. The range-finding test was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance in the final report, but the raw data of this test will be archived under the project number of the present study.
- Test concentrations: The concentrations tested were 100, 10, 1.0, 0.1 mg test item/L.
- Test Concentrations (Definitive Test): 0.1, 0.3, 1.0, 3.2, and 10 mg test item/L corresponding to the following geometric mean concentrations: 0.0667, 0.180, 0.693, 2.79 and 8.44 mg test item/L
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.7 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.:
0.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
4.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.067 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.639 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 96 hours of test duration did not show any difference between the algae that had been growing up to a test concentration of 0.639 mg test item/L and the algal cells in the control. From 2.79 onwards the algae showed deformations as the shape of the cells was rotund.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There were no remarkable observations related to the appearnce of the test item in the test media.
Results with reference substance (positive control):
A toxic reference (potassium dichromate) was tested to confirm the sensitivity of the test organisms in the test facility, as a separate study (Project No.: 110660210). The summary of the most recently performed toxic reference study are presented in Table 2). The results of this test are within the range of the historical data at the testing facility.
Reported statistics and error estimates:
Statistical Analysis: Based on the calculated cell densities, the 72 and 96-hour ErC50, EbC50 and EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis. For the determination of the 72 and 96-hour LOEC and the 72 and 96-hour NOEC, the calculated growth rates, biomass and yields at each test concentration were tested for significant differences compared to the control values by the Williams t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Table 1: Biological Results

 

72 hours

96 hours

Parameter

Biomass

Growth Rate

Yield

Biomass

Growth Rate

Yield

EC50[mg/L]

95% CI

1.3

1.2-1.4

3.5

3.1-3.8

0.92

0.88-0.98

1.2

1.1-1.3

4.2

3.9-4.5

1.3

1.2-1.3

EC20[mg/L]

95% CI

0.61

0.54-0.67

1.7

1.4-2.0

0.42

0.38-0.45

0.57

0.52-0.61

2.0

1.7-2.2

0.68

0.63-0.72

EC10[mg/L]

95% CI

0.41

0.35-0.46

1.2

0.9-1.5

0.28

0.24-0.31

0.39

0.35-0.43

1.3

1.1-1.6

0.49

0.44-0.53

NOEC [mg/L]

0.18

0.18

0.067

0.067

0.0639

<0.067

LOEC [mg/L]

0.69

0.69

0.18

0.180

2.79

0.067

n.d. = not determinable

Values refer to geometric mean measured test concentrations

Table 2:Results of the Most Recent Test withPseudokirchneriella subcapitata, renamed asRaphidocelis subcapitataperformed with the Referecne Item Potatssium dichromate (Study No.: 110660210)

Parameter                             (0-72 h)

Yield                                   [mg test item/L]

Growth rate                           [mg test item/L]

Biomass                           [mg test item/L]

72-hour EC50

95% conf. interval

0.402

0.373-0.434

0.963

0.927-1.001

0.414

0.385-0.446

72-hour EC20 

95% conf. interval

0.225

0.197-0.250

0.513

0.482-0.543

0.224

0.198-0.248

72-hour EC10

95% conf. interval

0.166

0.139-0.191

0.369

0.339-0.398

0.163

0.137-0.186

72-hour NOEC

0.094

0.094

0.094

72-hour LOEC

0.21

0.21

0.21

 values refer to nominal test concentrations

Validity Criteria of the Study

Cell Density Increase in Control Cultures: 318-fold increase within 72 hours and thus, validity criterion was met (at least by a factor of 16 within 72 hours).

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 22.7 % and thus, validity criterion was met (must not exceed 35 %).

Coefficient of Variation of Average Growth between Control Replicates: 0.8 % and thus, validity criterion was met (must not exceed 7 %).

Analytical Results

At the start of the test the recoveries of the test item ranged from 55 - 125 % of the nominal test concentrations (mean value of 75 %, all test concentrations considered). After 96 hours test duration, the recoveries ranged from 38 - 89 % of the nominal values (mean value of 73 %, all test concentrations considered). Since not all test concentrations were at ±20 % of the initial determined concentrations at test end, all reported results refer to geometric mean concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata, renamed as Raphidocelis subcapitata, was assessed in a static concentration-response test. The 96-hour ErC50 value was calculated to be 4.2 mg test item/L, the 96-hour EbC50was calculated to be 1.2 mg test item/L and the 96-hour EyC50 was calculated to be 1.3 mg test item/L. The 96-hour NOErC was determined to be 0.639 and the associated 96-hour LOErC was 2.79 mg/L. The 96-hour NOEbC was determined to be 0.067 and the associated 96-hour LOEbC was 0.180 mg/L. The 96-hour NOEyC was determined to be < 0.067 and the associated 96-hour LOEyC was 0.067 mg/L.
Executive summary:

1.1 Study Design

The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata, renamed as Raphidocelis subcapitata, over 96 hours under static conditions. The test was performed in accordance with OECD Guideline 201: "Freshwater Alga and Cyanobacteria”. Exponentially growing cultures of this unicellular green algal species were exposed to nominal concentrations of 10, 3.2, 1.0, 0.3. and 0.1 mg test item/L, alongside a control under defined conditions. Initial target cell density was 5000 cells/mL. The test was performed in 50 mL Erlenmeyer flasks under continuous illumination at 7240 to 7650 lux at cell culture level. The flasks contained as much test medium as possible and were kept close with a glass stopper to avoid loss of the test item due to volatilisation. After 24, 48, 72 and 96 hours, the cell growth was determined by spectrophotometric measurement. The mean value of the cell density was plotted versus time to produce growth curves for each concentration. Endpoints reported are the EC10, EC20, EC50, NOEC (No Observed Effect Concentration) and LOEC (Lowest Observed Effect Concentration) for growth rate, yield and area under the growth curve after 96 hours.

1.2 Results

At the start of the test the recoveries of the test item ranged from 55 - 125 % of the nominal test concentrations (mean value of 75 %, all test concentrations considered). After 96 hours test duration, the recoveries ranged from 38 - 89 % of the nominal values (mean value of 73 %, all test concentrations considered). Since not all test concentrations were at ±20 % of the initial determined concentrations at test end, all reported results refer to geometric mean concentrations.

 

72 hours

96 hours

Parameter

Biomass

Growth Rate

Yield

Biomass

Growth Rate

Yield

EC50[mg/L]

95% CI

1.3

1.2-1.4

3.5

3.1-3.8

0.92

0.88-0.98

1.2

1.1-1.3

4.2

3.9-4.5

1.3

1.2-1.3

EC20[mg/L]

95% CI

0.61

0.54-0.67

1.7

1.4-2.0

0.42

0.38-0.45

0.57

0.52-0.61

2.0

1.7-2.2

0.68

0.63-0.72

EC10[mg/L]

95% CI

0.41

0.35-0.46

1.2

0.9-1.5

0.28

0.24-0.31

0.39

0.35-0.43

1.3

1.1-1.6

0.49

0.44-0.53

NOEC [mg/L]

0.18

0.18

0.067

0.067

0.0639

<0.067

LOEC [mg/L]

0.69

0.69

0.18

0.180

2.79

0.067

n.d. = not determinable

Values refer to geometric mean measured test concentrations

1.3 Conclusion

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata, renamed as Raphidocelis subcapitata, was assessed in a static concentration-response test. The 96-hour ErC50 value was calculated to be 4.2 mg test item/L, the 96-hour EbC50was calculated to be 1.2 mg test item/L and the 96-hour EyC50 was calculated to be 1.3 mg test item/L. The 96-hour NOErC was determined to be 0.639 and the associated 96 -hour LOErC was 2.79 mg/L. The 96-hour NOEbC was determined to be 0.067 and the associated 96-hour LOEbC was 0.180 mg/L. The 96-hour NOEyC was determined to be < 0.067 and the associated 96-hour LOEyC was 0.067 mg/L.

Description of key information

Toxicity of isobutylamine to R. subcapitata was tested in an algal growth inhibition GLP test with closed vessel design according to OECD guideline 201. The 72-hour ErC50value was calculated to be 3.5 mg test item/L, the 72-hour EbC50was calculated to be 1.3 mg test item/L and the 72-hour EyC50was calculated to be 0.92 mg test item/L. The 72-hour NOErC was determined to be 0.18 and the associated 72-hour LOErC was 0.69 mg/L. The 72-hour NOEbC was determined to be 0.18 and the associated 72-hour LOEbC was 0.69 mg/L. The 72-hour NOEyC was determined to be 0.067 and the associated 72-hour LOEyC was 0.18 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
3.5 mg/L
EC10 or NOEC for freshwater algae:
1.2 mg/L

Additional information