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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:
From 18 February to 21 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations an frequency: Samples were taken from all test concentrations and the control at t=0h, t=24 h and t=72h.
- Sampling method: 1.0 mL from the approximate center of the test vessels. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 1.1 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sample storage conditions before analysis: Samples were stored in 2 mL True View Vials in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
Vehicle:
no
Details on test solutions:
The batch of Tributyl(ethyl) phosphonium diethylphosphate tested was a clear colourless liquid with a purity of 96.3% and was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L applying approximately 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E4 cells/mL.
Since it was expected that part of the test item adsorbed to glass surfaces during development of the analytical method (Test Facility Study No. 20175974), test vessels were pre-incubated with test solutions prior to the exposure phase in order to saturate the glass surface with test item, herewith minimizing the loss of test item from solution during the exposure period. Test solutions were added to the respective test vessels and were statically incubated under test conditions for an overnight period in the combined limit/range-finding test and for approximately three days in the final test. The period of pre-incubation in the final test was chosen for practical reasons and not intended for scientific purposes.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata),
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Pre-culture: 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use

ALGAE CULTURE
- Stock culture : Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C
- Culturing media and conditions: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Light Intensity : 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Between 22 and 23°C
pH:
From 7.7 to 8.3
Nominal and measured concentrations:
Nominal concentrations: 0.054, 0.24, 1.1, 4.9, 22 and 100 mg/L
Measured concentrations: The measured concentrations were at the level of nominal throughout the test, i.e. varied between 93 – 110% relative to nominal (see also Table 5 of the "Attached background material").
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation
- Material, size, headspace, fill volume: containing 50 mL of test solution.
- Renewal rate of test solution: none, static
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: Yes, test medium without test item or other additives
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 1 replicate of each test concentration without algae
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

TEST MEDIUM / WATER PARAMETERS
- Medium: M2
- pH: At the beginning and at the end of the test, for all test concentrations and the control.
- Temperature of medium: Continuously in a temperature control vessel.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the 1.1 mg/L test concentration to observe for any abnormal appearance of the algae compared to the control.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: Continuously illuminated using TLD-lamps
- Photoperiod, Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 78 to 84 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 4.5
- Range finding study: A combined limit/range-finding test was performed. Six replicates of exponentially growing algae were exposed to a control and a nominal concentration of 100 mg/L. In addition, three replicates per concentration were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: In the range finding study, inhibition of growth rate and yield increased with increasing concentration of the test item, resulting in 56% growth rate and 93% yield inhibition at the highest test concentration after 72 hours of exposure. The measured concentrations were at the level of nominal throughout the test, i.e. varied between 89 and 105% relative to nominal mg/L Based on these results the following concentrations were selected for the final test: 0.054, 0.24, 1.1, 4.9, 22 and 100 mg/L
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 3.4-4.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95%-CI: 1.12-1.20 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 0.53-0.91 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95%-CI: 0.39-0.50 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 1.1 mg/L when compared to the control.
- Other: A first full test was performed based on the results of the preceding combined limit/range-finding test. Since the coefficient of variation of the section-by-section specific growth rate in the control group exceeded 35% (i.e. 46%), the test was considered invalid and thus not further reported. Consequently, a second full, and also final test was performed with similar test procedures and conditions.
Additional details on the results are provided in section "Attached background material"
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh t-Test after Bonferroni-Holm correction, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding concentrations of the test item.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Tributyl(ethyl) phosphonium diethylphosphate
Nominal conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.772

0.0212

6

 

0.054

1.758

0.0087

3

0.83

0.24

1.771

0.0069

3

0.078

1.1

1.558

0.0296

3

12*

4.9

0.645

0.1340

3

64*

22

0.279

0.1078

3

84*

100

0.136

0.1608

3

92*

* Effect was statistically significant.

 

Table 2 : Yield And Percentage Inhibition For The Total Test Period

 Tributyl(ethyl) phosphonium diethylphosphate

Nominal conc. (mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

203.1

13.00

6

 

0.054

194.0

5.09

3

4.5

0.24

201.9

4.20

3

0.57

1.1

106.5

9.72

3

48*

4.9

6.3

2.88

3

97*

22

1.4

0.69

3

99*

100

0.6

0.82

3

100*

* Effect was statistically significant.


Validity criteria:

1.       In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 204).

2.       The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 5.9%).

3.       The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.2%).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Raphidocelis subcapitata, Tributyl(ethyl) phosphonium diethylphosphate reduced growth rate of this fresh water algae species statistically significantly at 1.1 mg/L and higher. The EC50 for growth rate inhibition (72h-ErC50) was 3.9 mg/L and the EC10 for growth rate inhibition (72h-ErC10) was 0.72 mg/L. The 72h-NOEC for both growth rate and yield inhibition was 0.24 mg/L, based on both statistical significance and biological relevance.

Executive summary:

A GLP compliant 72 hour static toxicity test with Raphidocelis subcapitata (formerly known asSelenastrum capricornutum and Pseudokirchneriella subcapitata) was performed in accordance with OECD 201 to assess the toxicity of Tributyl(ethyl) phosphonium diethylphosphate.

The final test was performed based on the results obtained in a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal test item concentrations of 0.054, 0.24, 1.1, 4.9, 22 and 100 mg/L (nominal concentrations). The initial algal cell density was 10E4 cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. All measured concentrations relevant for determination of effect parameters were at the level of nominal throughout the test, i.e. varied between 93 and 110% of the nominal concentrations. Effect parameters were therefore expressed in terms of analytically confirmed nominal concentrations. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, Tributyl(ethyl) phosphonium diethylphosphate inhibited growth rate and yield of this fresh water algae species significantly at analytically confirmed nominal concentrations of 1.1 mg/L and higher. The 72h-EC50 for growth rate inhibition (ErC50) was 3.9 mg/L with a 95% confidence interval ranging from 3.4 to 4.4 mg/L. The 72h-EC50 for yield inhibition was 1.20 mg/L with a 95% confidence interval ranging from 1.12 to 1.20 mg/L. The 72h-NOEC for growth rate and yield inhibition was 0.24 mg/L.

Description of key information

The toxicity of Tributyl(ethyl) phosphonium diethylphosphate to the freshwater green algae Raphidocelis subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. Statistically significant growth rate and yield inhibition was found at concentrations of nominally 1.1 mg/L and higher. Inhibition of growth rate and yield increased with increasing concentration of the test substance from nominally 1.1 mg/L upwards, resulting in 92% growth rate and 100% yield inhibition at the highest test concentration. The EC50 for growth rate inhibition (72h-ErC50) was 3.9 mg/L, the 72h-NOEC for both growth rate and yield inhibition was 0.24 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
3.9 mg/L
EC10 or NOEC for freshwater algae:
0.24 mg/L

Additional information