Phosphinylidynetrimethanol

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:

2018-04-16 - 2018-04-19 (experimental phase)

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:

"OECD guidelines for testing of chemicals 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test " (2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analyses were taken from freshly prepared solutions and after 72 hours at test conditions.
THPO was analysed by UHPLC-am-MS to demonstrate the integrity of the molecules during the test (qualitative evaluation): 2 µL samples were directly injected into the MS. During the test triplicate samples (10 ml) were collected from the freshly prepared blank solution and the highest and lowest test concentrations. 100 ml of these test solutions were also placed at normal test conditions (500 ml disposable test vials with air permeable stopper, 23 °C, 100 rpm, appr. 3000 lux) for 72 hours, and again samples were taken from all 3 solutions. Samples were stored in glass vials at -20 °C until measurement.
Total phosphorous (P) was analysed by High-resolution ICP-MS to quantify the THPO concentration: samples (5ml) were collected in parallel to those for the UHPLC measurements from the same solutions at the start and after 72 hours at test conditions. These samples were collected in polyethylene tubes containing HNO3 (final concentration 2 %). For the measurements, the test samples were diluted 1/10 or 1/100 in 2 % HNO3.

Vehicle:

yes

Remarks:

Algae OECD medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was dissolved in OECD medium directly.
- Controls: Algae OECD medium was used as a control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CODE CCAP: 278/4 - batch 3203/03
- Source (laboratory, culture collection): Test organisms are grown at the VITO laboratory facility.
- Age of inoculum (at test initiation): Algae that were used for the current test were transferred from the breeding medium to the OECD mineral medium on 13/04/2018 (4 days before the start of the test).
- Method of cultivation: Cultivation of the algae was performed using Jaworski medium as breeding medium. No further details on cultivation are specified.

ACCLIMATION
- Acclimation period: Algae that were used for the current test were transferred from the breeding medium to the OECD mineral medium on 13/04/2018 (4 days before the start of the test).
- Culturing media and conditions (same as test or not): Cultivation of the algae was performed using Jaworski medium as breeding medium. No further details on cultivation are specified.
- Any deformed or abnormal cells observed: not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no details given

Test temperature:

23°C ± 2°

pH:

7.89 - 8.05 at test start
9.35 - 10.38 at test end

Dissolved oxygen:

no details given

Salinity:

no details given

Conductivity:

no details given

Nominal and measured concentrations:

Nominal: 100 - 50 - 25 - 12.5 - 6.25 mg/l
Results showed that recovery at the end of the test is > 80% of the initial values for both analytical methods.
Based on the composition of the medium the expected P concentration in blank OECD medium is 360 µg/l P. In the test dilutions 1660 and 22360 µg/l P are expected in the 6.25 and 100 mg/l THPO dilutions respectively (THPO = C3H9O4P => 22 % P). The measured values are within the expected range (at least 89 % of the nominal value). It is concluded that the test item is stable during the test and test concentrations are comparable to the nominal values throughout the test period (at least 80 % recovery).

Details on test conditions:

TEST SYSTEM
- Test vessel: disposable 500 ml vessels
- Type (delete if not applicable): closed with air permeable stoppers
- Material, size, headspace, fill volume: fill volume: 100 ml medium or test dilution and 100 µl algal inoculum
- Aeration: no
- Initial cells density: appr. 5800 cells/ml
- No. of vessels per concentration (replicates): 3 for test dilutions
- No. of vessels per control (replicates): 6 for controls and 2 abiotic controls

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae OECD medium was used as a control and dilution medium (composition see "Any other information on materials and methods incl. tables")
- Culture medium different from test medium: Yes. Breeding medium is Jaworski medium, while OECD mineral medium was used for the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Temperature: 23°C ± 2° per test
- Adjustment of pH: not specified
- Photoperiod: continuous light
- Light intensity and quality: appr. 2650 lux
- Other: continuous shaking (100 rpm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily fresh samples (800 µl) were taken from each test vessel to calculate biomass.
- Determination of cell concentrations: At the start, a dilution series of the algae is measured both by Coulter counter (providing the number of cells in each dilution) and by Fluoroskan (providing a fluorescent signal). The correlation between both signals was then used during the rest of the test to recalculate the biomass from the Fluoroskan signal.
Daily fresh samples (800 µl) were taken from each test vessel and transferred to a 48 well plate. The fluorescence in each well was measured using a plate reader Fluoroskan and recalculated to the number of cells (biomass).
The mean fluorescent signal measured in the abiotic vessels is considered as blank signal and is used to correct fluorescent signals in the biotic compartments.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes. The preliminary range-finding test showed an EC50 value between 10 and 100 mg/l and NOEC/LOEC = 10/100 mg/l.
- Test concentrations: 100 - 50 - 25 - 12.5 - 6.25 mg/l THPO

Reference substance (positive control):

yes

Remarks:

Periodically an algal growth inhibition test is performed with ZnCl2 as a reference item. The last test was performed in February 2018.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not specified
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, results showed that recovery at the end of the test is > 80 % of the initial values for both analytical methods. Based on the composition of the medium the expected P concentration in blank OECD medium is 360 µg/l P. In the test dilutions 1660 and 22360 µg/l P are expected in the 6.25 and 100 mg/l THPO dilutions respectively (THPO = C3H9O4P => 22% P). The measured values are within the expected range (at least 89 % of the nominal value).

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the obtained result is within the historical range (0.08 - 0.17 mg/l: mean value 0.12 mg/l).
- EC50: EC50 (72h) = 0.10 mg/l ZnCl2

Reported statistics and error estimates:

In controls:
a) The biomass should increase at least 16x from 0-72 hours, i.e. a minimum value for a specific growth rate of 0.92/day. This was the case: biomass increased 88 x, specific growth rate was 1.49/day.
b) The coefficient of variance (CV) for the mean specific growth rate for the individual days (days 0-1, 1-2, 2-3) < 35 %. This was the case: CV was 19 %.
d) The coefficient of variance for the specific growth rate for day 0 - 3 for replicates < 7 %. This was the case: CV was 2 %.

Validity criteria fulfilled:

yes

Conclusions:

- ErC50 (72h): > 100 mg/l (nominal)
- EyC50 (72h): 99 mg/l (nominal)
- NOEC/LOEC (72h): 50/100 mg/l for both yield and growth rate (nominal)
- The test item THPO has a slight inhibitory effect on the algal growth rate and yield at the highest test concentration (100 mg/l, nominal).

Executive summary:

The aim of this GLP study was to evaluate the effect of the presence of the test item Tris(hydroxymethyl)phosphine oxide on the growth rate and yield of exposed green unicellular algae Pseudokirchneriella subcapitata. The acute toxicity test is based on the OECD test protocol "OECD guidelines for testing of chemicals 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test "(2011).

The study was performed with 5 nominal test concentrations: 100 - 50 - 25 - 12.5 - 6.25 mg/l Tris(hydroxymethyl)phosphine oxide, incubation time (test organism: Pseudokirchneriella subcapitata) was 72 hours.The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours. Validity criteria were met.

Results: The effect parameters were:

- ErC50 > 100 mg/l,

- EyC50 = 99 mg/l

- NOEC/LOEC= 50/100 mg/l for both yield and growth rate.

Based on the obtained results THPO does not need to be classified as hazardous to the environment, neither acute nor chronic.

Description of key information

- ErC₅₀ (72h) for freshwater algae (Pseudokirchneriella subcapitata): >100 mg/l [nominal; based on growth rate; OECD 201]

- NOEC (72h) for freshwater algae (Pseudokirchneriella subcapitata): 50 mg/l [nominal; based on both growth rate and yield; OECD 201]

- LOEC (72h) for freshwater algae (Pseudokirchneriella subcapitata): 100 mg/l [nominal; based on both growth rate and yield; OECD 201]

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

The aim of this GLP study was to evaluate the effect of the presence of the test item Tris(hydroxymethyl)phosphine oxide on the growth rate and yield of exposed green unicellular algae Pseudokirchneriella subcapitata. The acute toxicity test is based on the OECD test protocol "OECD guidelines for testing of chemicals 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test "(2011). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed with 5 nominal test concentrations: 100 - 50 - 25 - 12.5 - 6.25 mg/l Tris(hydroxymethyl)phosphine oxide, incubation time (test organism:Pseudokirchneriella subcapitata) was 72 hours.The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours. Validity criteria were met.

Results: The effect parameters were:

- ErC50 > 100 mg/l,

- EyC50 = 99 mg/l

- NOEC/LOEC= 50/100 mg/l for both yield and growth rate.

Based on the obtained results THPO does not need to be classified as hazardous to the environment, neither acute nor chronic.