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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:
19 Mar 2018 - 25 Mar 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)
Version / remarks:
2006 (corrected 2011)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4 11/07/00
Version / remarks:
Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
13 Mar 2017
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations
- Sampling method: duplicate samples at t=0 and t=72 h; additionally at t=24 and t=48 h for the additional vessels for analytical sampling.
- Sample storage conditions before analysis: Extracts were stored at 4 ± 4°C until analysis.
- The contents of the test beakers of each treatment were pooled at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A defined amount of the test item was added directly to the test water for each test concentration and was carefully stirred with 250 rpm for 24 hours in the dark to dissolve as much of the test item as possible. The highest nominal loading rate of 300 mg test item/L was prepared by mixing 315.3 mg test item into 1051 mL test water, for the nominal loading rate of 120 mg test item/L, 126.0 mg test item were mixed into 1050 mL test water, for the nominal loading rate of 48 mg test item/L, 50.1 mg were mixed into 1044 mL test water. The nominal loading rate of 19.2 mg test item/L was prepared by 46.5 mg test item were mixed into 2422 mL test water and for the nominal loading rate of 7.7 mg test item/L, 18.5 mg test item into 2400 mL test water. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration.
The test media were prepared just before introduction of the algae (= start of the test).
- Controls: test water without addition of the test item.
- There were no remarkable observations in the test media (clear test medium) at any point during the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG
- Source: „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
- Pre-culture of the algae: exponentially growing pre-culture, set up 4 days prior to the test start under the same conditions as in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
21.0 - 22.4 °C
pH:
At start: 8.1 - 8.3
At end: 8.7 - 9.4
Nominal and measured concentrations:
Nominal loading rates: 300, 120, 48, 19.2 and 7.7 mg test item/L (each prepared in independent WAF solutions) and a control
Measured concentrations: 62.8, 18.1, 6.34, 2.05 and 0.308 mg test item/L (geometric mean measured concentrations). Since the test item concentrations were not within ±20% of the measured initial concentrations during the test, effect parameters were based on geometric mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium.
- Aeration: no
- Initial cells density: 5000 cells/mL
- Control end cells density: 66 x 10^4 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Two additional replicates of each test item concentration and the control were set up for analytical dose verification after 24 and 48 hours.
- One additional replicate of each test concentration and of the control without algae were used to provide a 'blank' for the spectrophotometric measurements.
- Test vessels were continuously stirred with magnetic stirrers. The flasks were covered with glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.

GROWTH MEDIUM
- Standard medium used: yes, adjusted OECD medium (enriched with 350 mg NaHCO3/L)
- According to the Study Plan, OECD medium with 50.0 mg/L NaHCO3 would be used. However, the enriched test medium was taken by mistake. It was considered that this had no effect on the study, because the algal cells grew well and all validity criteria were fulfilled.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (OECD medium made with deionised water)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature: measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. pH: measured measured in all test item concentrations and the control at the start and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous, intensity: 6915 lux (range: 6560 - 7270 lux) measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: 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 and 72 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.

TEST CONCENTRATIONS
- Range finding study: Pre-experiments were performed (non-GLP) to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed July 2017)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 21.3-22.2
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 10.8-11.9
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.308 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a geometric mean measured concentration of 62.8 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h ErC50: 1.02 mg/L (95% confidence interval: 0.984 - 1.05 mg/L)
Reported statistics and error estimates:
Calculation of ErCx values: Probit analysis.
NOEC determination: Dunnett’s t-test, alpha = 0.05, one-sided
Statistical analysis was performed with ToxRat Professional, Version 3.2.1, ToxRat(r) Solutions GmbH.

Table 1. Summary of Analytical Results

Sample description

Nominal

Geometric mean2

concentration

% of

Concentration3

[mg test item/L]

Nominal1

[mg test item/L]

Control

<LOD

n.a.

7.7

4

0.308

19.2

11

2.05

48

13

6.34

120

15

18.1

300

21

62.8

1Mean value of all measured samples per treatment group
2Calculated according to OECD Guidance Document No. 23, Annex 2
3The tabulated results represent results rounded to three significant digits

n.a.: not applicable

Table 2: Growth rates µ and percentage inhibition of µ during the Test Period

Geometric Mean Measured Concentration [mg test item/L]

Growth rates µ [1/day] and % inhibition of µ

0 – 24 hours

0 – 48 hours

0 – 72 hours

µ

%

µ

%

µ

%

Control

1.163

-

1.503

-

1.627

-

0.308

1.056

9.1

1.481

1.4

1.597

1.9

2.05

1.002

13.8

1.355

9.8

1.548

4.9 *

6.34

1.244

-7.0

1.301

13.4

1.544

5.1 *

18.1

0.000

100.0 *

0.322

78.6

1.049

35.5 *

62.8

0.000

100.0 *

0.048

96.8

0.000

100.0 *

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control
(tested withDunnett’s t-test (24, 48 and 72h),a = 0.05, one-sided)

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 72-hour ErC50 value was calculated to be 21.7 mg test item/L, based on geometric mean measured concentrations. The 72-hour ErC10 value was calculated to be 11.4 mg test item/L. The 72-hour NOErC was determined to be 0.308 mg test item/L.
Executive summary:

The toxicity to freshwater green aquatic algae was examined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, cultures of algae (P. subcapitata) were exposed to WAFs individually prepared at loading rates of 0 (control), 7.7, 19.2, 48, 120, and 300 mg/L for 72 hours. Nominal target concentrations were analytically verified, and geometric mean measured exposure concentrations determined to be 0.308, 2.05, 6.34, 18.1, and 62.8 mg/L, and effect concentrations expressed as such. Cell concentrations were determined at 24, 48, and 72 hours after the start of the test and inhibition of growth rate calculated. The test is valid.

A concentration-dependent and statistically significant inhibition of growth rate was observed from a test concentration of 2.05 mg/L onward with percentages inhibition calculated to be 4.9, 5.1, 35.5, and 100%. Microscopic examination revealed that algal cells shape was not obviously affected up to the highest tested concentration. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be 21.7 mg/L, and 11.4 mg/L, respectively. The 72-h NOErC is 0.308 mg/L.

Description of key information

The toxicity to freshwater green aquatic algae was examined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, cultures of algae (P. subcapitata) were exposed to WAFs individually prepared at loading rates of 0 (control), 7.7, 19.2, 48, 120, and 300 mg/L for 72 hours. Nominal target concentrations were analytically verified, and geometric mean measured exposure concentrations determined to be 0.308, 2.05, 6.34, 18.1, and 62.8 mg/L, and effect concentrations expressed as such. Cell concentrations were determined at 24, 48, and 72 hours after the start of the test and inhibition of growth rate calculated. The test is valid.

A concentration-dependent and statistically significant inhibition of growth rate was observed from a test concentration of 2.05 mg/L onward with percentages inhibition calculated to be 4.9, 5.1, 35.5, and 100%. Microscopic examination revealed that algal cells shape was not obviously affected up to the highest tested concentration. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be 21.7 mg/L, and 11.4 mg/L, respectively. The 72-h NOErC is 0.308 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
21.7 mg/L
EC10 or NOEC for freshwater algae:
11.4 mg/L

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