Phenylphosphonic acid

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:

11 Sep 2017 to 10 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pathlength of the immersion probe used to determine cell densities was 10 mm. Evaluation: This has no impact on the results of the analysis.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenylphosphonic acid

EC Number:

216-388-1

EC Name:

Phenylphosphonic acid

Cas Number:

1571-33-1

Molecular formula:

C6H7O3P

IUPAC Name:

phenylphosphonic acid

Test material form:

solid: particulate/powder

Details on test material:

Lot number LKF6065

Specific details on test material used for the study:

Appearance: White crystalline powder
Batch: LKF6065
Test item storage: At room temperature protected from light
Stable under storage conditions until: 09 June 2018 (expiry date)

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Acquity UPLC system (Waters, Milford, MA, USA)

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

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 the highest 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:

not specified

Details on test solutions:

The test material was a white crystalline powder with a purity of 100% and 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 a 14-minute period of magnetic stirring to accelerate dissolution of the test item in medium. The pH of the test solutions were adjusted from 8.2 (blank medium) and 4.1 (100 mg/L) to approximately 6.5 with 1M HCl and 1M NaOH (Merck, Darmstadt, Germany), respectively. 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 10 cells/mL.

Any residual volumes were discarded.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST SYSTEM
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae 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

Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater
purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

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 10 to the power of 4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

At the end of the test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

Test conditions

Hardness:

Pre-culture medium: Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C

pH:

The pH of the test solutions were adjusted from 8.2 (blank medium) and 4.1 (100 mg/L) to approximately 6.5 with 1M HCl and 1M NaOH (Merck, Darmstadt, Germany), respectively.

Phenyl Phosphonic Acid
concentration (mg/L) pH Levels Recorded
t=0h* t=72h
Control 8.2 → 6.7 7.5
100 4.1 → 6.6 6.9

Nominal and measured concentrations:

The results will be based on the nominal test item concentrations if the analytical program has confirmed that the measured test item concentrations remained within 20 % of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic

Fresh Water 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.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse
osmosis; Millipore Corp., Bedford, Mass., USA)

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 10 4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Test Concentrations
Phenyl Phosphonic Acid: 0.10, 1.0, 10 and 100 mg/L
Controls: Test medium without test item.
Replicates: 6 replicates of the control and the highest concentration,
3 replicates of each of the lower test concentrations,
1 extra replicate of each test group for sampling purposes,
1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10 to the 4cells/mL.
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.

i

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

based on statistical significance

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

based on biological relevance.

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

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

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

based on statistical significance

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

based on biological relevance.

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

No significant inhibition for both growth rate and yield was observed at the three lowest concentrations tested. An inhibition of 2.4% and 12% was observed at the highest concentration tested for growth rate and yield, respectively.

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 (student-t test for homogeneous variances, α=0.05, one-sided, smaller).

No EC50-values could be calculated (EC 50 > maximum concentration tested).

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Phenyl Phosphonic Acid reduced growth rate and inhibited the yield of this fresh water algae species statistically significantly at 100 mg/L. The NOEC for growth rate was nevertheless set at 100 mg/L as the inhibition of 2.4% was not considered biologically relevant. The NOEC for yield inhibition was below 100 mg/L based on both statistical significance and biological relevance.

Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

The objective of the study was to evaluate Phenyl Phosphonic Acid for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10 and EC50 for 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.

The batch of Phenyl Phosphonic Acid tested was a white crystalline powder with a purity of 100% and completely soluble in test medium at the concentrations tested.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to 100 mg/L in the limit test. In addition, three replicates per group were exposed to 0.10, 1.0 and 10 mg Phenyl Phosphonic Acid per litre in a combined range-finding test. The initial algal cell density was 10 to the power of 4 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.  

No significant inhibition for both growth rate and yield was observed at the three lowest concentrations tested. An inhibition of 2.4% and 12% was observed at the highest

concentration tested for growth rate and yield, respectively.

At the start of the test, the actual test concentrations were slightly above nominal. However, the same was true for the QC samples. At the end of the 72-hour period the measured concentration had not decreased by more than 20% in samples taken from the highest test concentration with algae. Results were therefore based on analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)         NOEC*       NOEC**       EC10              EC20              EC50

Growth rate                                   Value                       <100           100               >100              >100            >100

   Yield                                          Value                      <100            <100            >10<100        >100            >100

* - based on statistical significance, ** - based on biological relevance.