Benzyltriphenylphosphonium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 2017 to March 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Analytical purity: >=99.5%
- Purity test date: 02 March 2017
- Lot/batch No.: 180739
- Expiration date of the lot/batch: Not specified
- Appearance: Off-white powder
- Storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control
- Frequency At t=0 h, t=24 h and t=72 h
- Volume 2.0 mL
- Storage Samples were stored in a freezer (≤-15°C) until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of test item was an off-white powder with a purity of 100% that appeared not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying a 3 day period of magnetic stirring to ensure maximum dissolution of the test item in test medium (M2). Thereafter, the aqueous Saturated Solution (SS) was filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS 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 10000 cells/mL. No correction was made for the purity/composition of the test item.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: 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

CULTURE
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Pre-culture: 3 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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

At the end of the final test, microscopic observations of the appearance of the cells were performed on the control and the test solution containing 0.22% of the SS

Hardness:

24 mg CaCO3/L

Test temperature:

Continuously measured in a temperature control vessel: 22-23°C

pH:

Measured at the beginning and end of the test:
- Control: 8.5 (t=0h) / 8.2 (t=72h)
- Test concentrations: 8.2-8.3 (t=0h) / 8.0-8.2 (t=72h)

Nominal and measured concentrations:

Nominal concentrations (combined limit/range-finding test): 1.00 - 10.00 - 100.00% of the SS
Nominal concentrations (additional range-finding test): 0.01 - 0.10 - 1.00% of the SS
Nominal concentrations (final test): 0.10 - 0.22 - 0.46 - 1.00 - 2.20% of the SS
Controls: Test medium without test item or other additives

Details on test conditions:

TEST SYSTEM (final test)
- Replicates:
3 replicates of each test concentration
6 replicates of the control
1 or 2 replicates of each test concentration without algae
1 or 2 replicates of each test concentration and the control for sampling after 24 hours
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: Initial cell density of 10000 cells/mL. 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 (pathlength =10 mm). Algal medium was used as blank
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 96 to 97 µE.m-2.s-1
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.56-0.61 mg/L (95% confidence interval)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.22-0.25 mg/L (95% confidence interval)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.22-0.27 mg/L (95% confidence interval)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.086 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.074-0.097 mg/L (95% confidence interval)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h-ErC50: 0.99 mg/L (range for expected responses: 0.82-2.3 mg/L)

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 test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.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 analytically confirmed nominal concentrations of the test item.

Combined Limit/Range-Finding Test

Growth rates of algae exposed to the test solution containing 1% of the SS were inhibited by 84% at the end of the exposure period. Consequently, the EC50 was expected to be lower than the lowest concentration tested. Based on these results, samples for determination of actual exposure concentrations were not analysed and it was decided to perform an additional range‑finding test. All test conditions were maintained within the limits prescribed by the study plan.

Additional Range-Finding Test

The expected EC50 was between solutions containing 0.10 and 1.0% of the SS prepared at loading rate of 100 mg/L. Based on these results samples taken from the solutions containing 0.010 and 1.0% of the SS were analysed. The initial concentrations were 0.029 and 0.91 mg/L at the start of the test, respectively. These concentrations remained stable during the test period (102-107% of initial). It should be noted that the concentrations measured in the samples collected from the test solution containing 0.010% of the SS, which were at an unrealistic level of 290-310% of nominal throughout the test duration, were extrapolated from the calibration curve since they were below the limit of quantification. Additionally, the mean accuracy for the quality control samples containing test item at target concentration of 0.1 mg/L was above the acceptability criteria of 70-110% (i.e. the mean accuracy was 126%) and, consequently, measured concentrations have to be considered as estimated values. All test conditions were maintained within the limits prescribed by the study plan.

Final Test

Samples taken from all the test concentrations were analysed. The measured concentration were in agreement with nominal, despite the filtration, throughout the test duration (100-108%), with the exception of the concentrations measured in the samples collected from the highest test concentration (i.e, solution containing 2.2% of the SS), which were at the level of 50% of nominal throughout the test duration. Based on the water solubility provided by the Sponsor (i.e., 30 g/L – SDS data), it was excluded that the maximum solubility of the test item in test medium was already reached in the test solution containing 1% of the SS. The reason for the observed result remained unclear. Consequently, it was decided to exclude the responses recorded at the highest test concentration from the analysis of the data and to base the effects parameters on the analytically confirmed nominal concentrations of the remaining test concentrations. Additionally, it was concluded that any undissolved material filtered during preparation of the test solutions was not test item related.

Growth rates were increasingly reduced with increasing concentrations of the test item from 0.10 mg/L upwards, resulting in ≥80% inhibition at the two highest test concentrations (Table 1). Statistically significant inhibition of growth rate was found at all test concentrations. However, the effect observed at the lowest test concentration was biologically not relevant, i.e. it was <10%. Inhibition of yield increased with increasing concentration of the test item, resulting in ≥99% inhibition at the two highest test concentrations (Table 2). Statistically significant inhibition of yield was found at all test concentrations. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.22 mg/L when compared to the control.

Table 1. Growth rate and percentage inhibition for the total test period

Nominal concentration of test item (mg/L)	Mean growth rate (day^-1)	Std. dev.	n	Percentage inhibition (%)
Control 0.10 0.22 0.46 1.00 2.20*	1.774 1.713 1.580 1.190 0.352 0.016	0.0209 0.0236 0.0597 0.0770 0.0500 0.0286	6 3 3 3 3 3	3.5*** 11.0** 33.0** 80.2** 99.1

* Statistical analysis were not conducted on this test concentration. ** Effect was statistically significant. *** Effect statistically significant but biologically not relevant (<10%).

Table 2. Yield and percentage inhibition for the total test period

Nominal concentration of test item (mg/L)	Mean yield (x10^4 cells/mL)	Std. dev.	n	Percentage inhibition (%)
Control 0.10 0.22 0.46 1.00 2.20*	204.4 169.7 114.7 35.1 1.9 0.1	12.78 11.83 20.58 8.04 0.45 0.09	6 3 3 3 3 3	17.0** 43.9** 82.8** 99.1** 100.0

* Statistical analysis were not conducted on this test concentration.** Effect was statistically significant.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the test item reduced the growth rate of the freshwater green algae Pseudokirchneriella subcapitata at analytically confirmed nominal concentrations of 0.22 mg/L and higher, based on biological relevance. The EC50 for growth rate inhibition (72h-ERC50) was 0.59 mg/L with a 95% confidence interval ranging from 0.56 to 0.61 mg/L. The EC10 for growth rate inhibition (72h-ERC10) was 0.25 mg/L with a 95% confidence interval ranging from 0.22 to 0.27 mg/L. The 72h-NOEC for growth rate inhibition was 0.10 mg/L, based on biological relevance.

Executive summary:

The toxicity of the test item to the freshwater green algae Pseudokirchneriella subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. The test item reduced the growth rate of the algae species at analytically confirmed nominal concentrations of 0.22 mg/L and higher, based on biological relevance. The EC50 for growth rate inhibition (72h-ERC50) was 0.59 mg/L with a 95% confidence interval ranging from 0.56 to 0.61 mg/L. The EC10 for growth rate inhibition (72h-ERC10) was 0.25 mg/L with a 95% confidence interval ranging from 0.22 to 0.27 mg/L. The 72h-NOEC for growth rate inhibition was 0.10 mg/L, based on biological relevance.

Description of key information

The toxicity of the test item to the freshwater green algae Pseudokirchneriella subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. The test item reduced the growth rate of the algae species at analytically confirmed nominal concentrations of 0.22 mg/L and higher, based on biological relevance. The EC50 for growth rate inhibition (72h-ERC50) was 0.59 mg/L with a 95% confidence interval ranging from 0.56 to 0.61 mg/L. The EC10 for growth rate inhibition (72h-ERC10) was 0.25 mg/L with a 95% confidence interval ranging from 0.22 to 0.27 mg/L. The 72h-NOEC for growth rate inhibition was 0.10 mg/L, based on biological relevance.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.59 mg/L

EC10 or NOEC for freshwater algae:

0.25 mg/L

Additional information

The toxicity of the test item to freshwater green algae Pseudokirchneriella subcapitata was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.