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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:
2017-03-03 to 2017-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0 (Sample code: NC), 0.5 (Sample code: A), 1.5 (Sample code: B), 4.5 (Sample code: C), 13.5 (Sample code: D), 40.5 (Sample code: E), 121.5 (Sample code: F) mg/L
- Sampling method: test solutions (NC, A, B, C, D, E and F, samples of 6 mL) were sampled and divided daily (0, 24, 48 and 72 h). Of each sampled treatment, one of the samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site menal GmbH and analysed.

The samples were filled into 10 mL glass vials. The sampling was conducted according to the following specification:
• At the start of the test (0 h), the test solutions NC, A, B, C, D, E and F were sampled before division to the test vessels (each group: 4 samples of 6 mL).
• After 24 h and 48 h exposure, both additional vessels of NC, A, B, C, D, E and F were sampled (one sample per replicate: 2 samples of 6 mL per group).
• At the end of the test (72 h), both additional vessels and 2 replicates of the test vessels from NC, A, B, C, D, E and F were sampled (one sample per replicate: 4 samples of 6 mL per group).
Of each sampled treatment, one of the samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site menal GmbH and analysed.

- Sample storage conditions before analysis: stored at ≤ -18 °C until measuring at the following day (48 h exposure).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 135.2 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) was added to 1000 mL test medium and shaked for 2.5 h using an overhead shaker at 22.4 °C in the dark. This stock solution was used as highest test item concentration in the test. The negative control (NC; test medium) was treated in the same way as the test item solution. Into each test vessel, 50 mL test item solution and test medium was transferred, respectively. The further test item concentrations were prepared by diluting the stock solution with test medium.
- Differential loading: nominal loading rates of 0, 0.5, 1.5, 4.5, 13.5, 40.5, 121.5 mg/L.
- Controls: yes

See also Table 1 "Preparation of the test treatment and control" in "Any other informations on materials and methods".
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen, subcultured at Hydrotox GmbH since May 2016
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Suspension culture in Holm-Hansen medium under axenic conditions

ACCLIMATION
-Before the start of the test, 1.25 mL of the algae stock suspension was diluted with 48.75 mL OECD TG 201 medium to obtain a concentration of 5 x 10^4 cells/mL. This pre-culture was incubated for 4 d at 22.3 - 22.5 °C and 70.7 μE/m²s +/- 5.3% continuous lighting. The cell concentration was determined using the Coulter Counter, resulting in 87.949 x 10^5 cells/mL. To obtain a nominal inoculum concentration of 7 x 10^4 cells/mL, 2.390 mL pre-culture was diluted into 300 mL OECD TG 201 medium.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.3 - 22.6 °C
pH:
7.3 - 7.7 in the control, 6.8 - 7.8 in the test item
see also Table 2 "pH at the start and the end of the test" in "Any other informations on materials and methods" below.
Nominal and measured concentrations:
Test item concentration nom. [mg/L]: 0, 0.5, 1.5, 4.5, 13.5, 40.5, 121.5
Test item concentration measured [mg/L]: 0, 0.2, 0.26, 0.40, 0.57, 1.67, 39.27
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks, Schott, Mainz
- Fill volume: 50 mL
- Aeration: no
- Initial cells density: 7 x 10^4 cells/mL
- Control end cells density: 5.437 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Medium used: Holm-Hansen medium

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: 70.7 μE/m²s +/- 5.3%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Counter Z2, Beckman Coulter, Krefeld
- Chlorophyll measurement: 96-well micro-plates, No. 655 101, Greiner bio-one, Frickenhausen; Fluorescence Microplate Reader, Tecan infinite F200, Tecan Group Ltd. Männedorf, Switzerland

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.21-0.67 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.10 - 0.27 mg/L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
<= 0.2 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.:
< 0.2 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): no abnormalities observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.99 mg/L (CL 95%: 0.89 - 1.10 mg/L)
- EyC50: 0.52 mg/L (CL 95%: 0.38 - 0.74 mg/L)
Reported statistics and error estimates:
With the statistical software ToxRat 3.2.1 (ToxRat Solutions GmbH, Alsdorf), ECx and LOEC/NOEC for yield and growth rate were determined.

Table 3: Analytically measured test item concentrations after 0 h, 24 h, 48 h and 72 h exposure.

Nominal test item concentration [mg/L] Test item concentration [mg/L]
(sample name)
Geometric mean [mg/L]*
0 h 24 h 48 h 72 h 0 h - 72 h 
NC N.D.  N.D. N.D. N.D. N.D.
0.5 0.19 N.D. N.D. N.D. 0.2
1.5 0.61 N.D. N.D. N.D. 0.26
4.5 3.05 N.D. N.D. N.D. 0.4
13.5 13.3 N.D. N.D. N.D. 0.57
40.5 48.7 4.02 N.D. N.D. 1.67
121.5 132 55.9 18.2 17.7 39.27
N.D.: Not Detected
*For measurements which are < LOQ, the value of LOQ, i.e. 0.2 mg/L, was used for calculation.

Table 4: Overview of the Effect concentrations based on Specific Growth Rate and Yield.

 

Specific Growth Rate
(72 h,
Geometric mean test item concentration [mg/L])

Yield

(72 h,Geometric mean test item concentration [mg/L])

EC50

0.37 mg/L

0.22 mg/L

EC20

0.22 mg/L

0.16 mg/L

EC10

0.17 mg/L

0.14 mg/L

Slope of the curve

0.0006

LOEC

≤ 0.20 mg/L

≤ 0.20 mg/L

NOEC

< 0.20 mg/L

< 0.20 mg/L

Validity criteria fulfilled:
yes
Conclusions:
For the static algae growth inhibition test conducted with Pseudokirchneriella subcapitata, applying nominal concentrations of of 0 (control), 0.5, 1.5, 4.5, 13.5, 40.5, 121.5 mg/L propyl gallate, results based on growth rate after 72 h were as follows: EC50: 0.37 mg/L (95 % C.I. 0.21 – 0.67 mg/L); EC20: 0.22 mg/L (95 % C.I. 0.14 – 0.35 mg/L); EC10: 0.17 mg/L (95 % C.I.0.10 – 0.27 mg/L) LOEC: ≤ 0.20 mg/L; NOEC: < 0.20 mg/L. As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.
Executive summary:

In a 72 hour algal growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to propyl gallate at nominal concentrations of 0 (control), 0.5, 1.5, 4.5, 13.5, 40.5, 121.5 mg/L and measured concentrations of 0, 0.2, 0.26, 0.4, 0.57, 1.67 and 39.27 mg/L (geometric mean) under static conditions in accordance with the OECD Guideline 201 (23 March 2006), OECD 23 (2000) and under GLP. The % growth inhibition in the treated algal culture as compared to the control ranged from 8.7 % at a concentration of 0.20 mg/L to 73.7 % at a concentration of 39.27 mg/L.

There were no compound related phytotoxic effects.  

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata (Strain No. 61.81 SAG)

Test Type: Static, 

As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.

Specific Growth Rate:

72 hr EC50: 0.37 mg/L                  95 % C.I. 0.21 – 0.67 mg/L;

72 hr EC20: 0.22 mg/L                  95 % C.l. 0.14 – 0.35 mg/L;

72 hr EC10: 0.17 mg/L                  95 % C.l. 0.10 – 0.27 mg/L;

72 hr NOEC: < 0.20 mg/L.  

 

Endpoint(s) Effected:  Specific growth rate, yield

Description of key information

The lowest valid 78-hr ErC50 is determined to be 0.37 mg/L by Schwartz (2017) in a GLP guideline study conforming to OECD 201.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition test conducted with Pseudokirchneriella subcapitata.

Furthermore, one publication reported by Zurita et al. (2007) is available, where the effect of propyl gallate to C. vulgaris was determined in a 72-hour acute toxicity test similar to OECD guideline 201/EU Method C.3.

The 72-hr ErC50 (growth rate) was determined to be 146.42 mg/L (nominal concentration).

Since detailed information on the test item or study system are missing and only nominal concentrations are used although the test item is susceptible to hydrolysis, this study is regarded methodologically deficient hence not suitable for the environmental hazard assessment of propyl gallate. Consequently, this toxicity study is disregarded.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
0.37 mg/L
EC10, LC10 or NOEC for freshwater algae:
0.17 mg/L

Additional information