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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 october 2017 to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Lot number: 2000850599
Analysis date: 19 August 2016
Specific details on test material used for the study:
Identification: Tri-o-tolylphosphine
Appearance: Off-white crystalline powder (determined by
Charles River Den Bosch)
Batch: B64002 (Pfizer Lot no. 2000850599)
Purity/Composition: 99%
Test item storage: At room temperature
Stable under storage conditions until: Unknown
CAS number: 6163-58-2

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from the SS and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.

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 at the analytical laboratory of the Test Facility.
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 an intermediate item concentration 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.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Tri-o-tolylphosphine tested was an off-white crystalline powder with a purity of 99.9% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the Saturated Solution (SS) was collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the test concentration. 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 104 cells/mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
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.
60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
M1 Media; 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)
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. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21-24°C
pH:
7.9-8.0
Nominal and measured concentrations:
A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the test concentration (nominal) which resulted in a TWA of 0.011 mg/L
Details on test conditions:
At the start of the test, the measured concentration in the SS with algae was 0.011 mg/L and the measured concentration in the SS without algae was similar. After 24 hours of exposure the measured concentration in the SS with algae was higher than the initial concentration in both the first and the reserve sample indicating that the test item concentration was above the solubility limit in test medium. The measured concentration in the SS without algae was below the limit of detection of the analytical method (i.e. below 0.0055 mg/L). After 72 hours of exposure the measured concentrations in the SS with and without algae were below the limit of detection of the analytical method.
Based on these results, the TWA concentration was calculated to be 0.011 mg/L, and used to determine the effect parameters. Note that the results of the measurement of the reserve sample for the 24h time period was used for the calculation of the TWA because this value was the lower of the two and therefore worst case.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.011 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
not determinable
Remarks:
At or greater than solubilty limit
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 0.011 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
not determinable
Remarks:
At or greater than solubilty limit
Details on results:
No significant differences were recorded between the values for growth rate or yield at the TWA concentration when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration when compared to the control.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded showed a stimulation rather than an inhibition of growth. No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).

Any other information on results incl. tables

The mean cell densities measured during the combined limit/range-finding test are presented inTable1.Table2andTable3present the percentages growth rate inhibition and yield inhibition per concentration, respectively. No inhibition of growth was found at any of the test concentrations and the control during the test period. However, the mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35% (i.e. was 36%).Therefore, the test was not valid.

Samples taken during the test were not analysed. All test conditions were maintained within the limits prescribed by the study plan.

Table 1

Mean Cell Densities (x104Cells/mL) during the Combined Limit/Range-Finding Test

Time (h) 

Tri-o-tolylphosphine, %SS at 100 mg/L

Control

1.0

10

100

0

1.0

1.0

1.0

1.0

24

3.0

n.m.

n.m.

6.1

48

30.7

n.m.

n.m.

38.4

72

176.8

184.3

181.8

194.8

n.m. not measured


Table2          
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

Tri-o-tolylphosphine, %SS at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.724

0.0208

6

1.0

1.739

0.0127

3

-0.8

10

1.734

0.0167

3

-0.6

100

1.757

0.0122

6

-1.9

 

Table3          
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

Tri-o-tolylphosphine, %SS at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

175.8

11.03

6

1.0

183.3

7.09

3

-4.2

10

180.8

9.16

3

-2.8

100

193.8

7.09

6

-10.2

Growth Rate and Percentage Inhibition for the Total Test Period

Tri-o-tolylphosphine, TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.950

0.0177

6

0.011

1.957

0.0167

6

-0.4



Growth Rate and Percentage Inhibition at Different Time Intervals

Tri-o-tolylphosphine,

TWA conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.334

 

1.946

 

1.570

 

0.011

6

2.480

-6.3

1.948

-0.1

1.442

8.1


 
Yield and Percentage Inhibition for the Total Test Period

Tri-o-tolylphosphine, TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

346.6

18.18

6

0.011

353.9

17.47

6

-2.1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, due to the low solubility of Tri-o-tolylphosphine in test medium, concentration levels that might induce significant inhibition of algal growth could not be reached.
The 72h-NOEC and the 72h-EC50 for inhibition of both growth rate and yield were at and above the concentration obtained in a SS prepared at 100 mg/L. The TWA concentration in this SS was 0.011 mg/L and this concentration was considered to be above the solubility limit of Tri-o-tolylphosphine in test medium.
Executive summary:

The objectiveofthe study was to evaluate Tri-o-tolylphosphine for its ability to generate toxic effects inPseudokirchneriella subcapitataduring an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Tri-o-tolylphosphine tested was an off-white crystalline powder with a purity of 99.9% and not completely soluble in test medium at the loading rate initially prepared.

A limit test was performed based on the results of a preceding combined limit/range-finding test. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the test concentration.

Six replicates of exponentially growing algal cultures per group were exposed to a control and to the SS. The initial algal cell density was 104cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the measured concentration in the SS was 0.011 mg/L. After 24 hours of exposure the measured concentration was higher than the initial concentration in both the first and the reserve sample indicating that the test item concentration was above the solubility limit in test medium. After 72 hours of exposure the measured concentration was below the limit of detection of the analytical method (i.e. below 0.0055 mg/L). Based on these results, the TWA concentration was calculated to be 0.011 mg/L and used to determine the effect parameters. No significant differences were recorded between the values for growth rate or yield at the TWA concentration when compared to the control group. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, due to the low solubility of Tri-o-tolylphosphine in test medium, concentration levels that might induce significant inhibition of algal growth could not be reached. The 72h-NOEC and the 72h-EC50for inhibition of both growth rate and yield were at and above the concentration obtained in a SS prepared at 100 mg/L.The TWA concentration in this SS was 0.011 mg/L and this concentration was considered to be above the solubility limit ofTri-o-tolylphosphinein test medium.