2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide

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 April 26, 2004 to May 28, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): PARAD Substance 139.
- Molecular formula: C6H10O3.xH3O4P.
- Molecular weight: ~250.
- CAS Number 52628-03-2.
- Description: Slightly yellowish viscous liquid.
- Batch: 31104251.
- Purity: n.a. (mixture).
- Test substance storage: At room temperature in the dark.
- Stability under storage conditions: Stable
- Expiry date: December 31, 2004
- Density: 1.4488 (determined at NOTOX)
- Stability in vehicle: Water -No, Acetone -Not indicated.

Analytical monitoring:

yes

Details on sampling:

During the final test singular samples for possible analysis were taken from all test concentrations and the controls.
- Frequency at t=0 h, t=24 h and t=72 h
- Volume 10 mL
- Storage Samples were stored in a freezer until analysis.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 h of exposure and at the end of the test period. Additionally, singular reserve samples of 10 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Details on test solutions:

Preparation of test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as
possible (e.g. film of the test substance on the water surface).

The batch of the substance tested was a slightly yellowish viscous liquid and was completely soluble in test medium at the concentrations tested.
Preparation of test solutions for the range-finding test started with a stock solution of 100 mg/L applying 20 mins of magnetic stirring to accelerate the dissolving of the test substance in the test medium. For the final test a stock solution of 320 mg/L was prepared applying 25 mins of
magnetic stirring followed by a 5-min treatment period with ultrasonic waves. The test concentrations were prepared by subsequent dilutions of the stock in test medium.

The final test solutions were all clear and colourless. Due to the acidic character of the test substance an additional control was prepared in both the range-finding and final test with the same pH level as the highest concentration tested to correct for possible pH-related effects on algal growth.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

other: Selenastrum capricornutum, strain: NIVA CHL 1.

Details on test organisms:

TEST ORGANISM
- Species: Selenastrum capricornutum, strain: NIVA CHL 1.
- Source: In-house laboratory culture.
- Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- 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 23±2°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature of the test medium was 22.3°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 23.6 and 23.8°C. Temperature remained within the limits prescribed by the protocol (temperature: 21- 25°C, constant within 2°C).

pH:

The pH was generally maintained within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit) except at 160 mg/L, which was a consequence of the acidic character of the test substance.

Nominal and measured concentrations:

10, 20, 40, 80 and 160 mg/L (nominal)

Details on test conditions:

- Controls: Test medium without test substance or other additives (blank-control) and one control with pH adjusted to the level of that measured in the highest test concentration (treatment-control).
- Replicates: 3 replicates of each test concentration, 6 replicates of the blank-control, 6 replicates of the treatment-control, 2 replicates of the highest concentration without algae, 1 extra replicate of each test concentration for sampling after 24 hours of exposure.

Test procedures and conditions
- Test duration: 72 h.
- Test type: Static.
- Test vessels: 100 mL, all-glass, containing 50 mL of test medium.
- Medium: M2.
- Cell density: An initial cell density of 1 x 10^4 cells/mL.
- Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 70 to 102 μE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator.
- During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate) in a separate study (NOTOX Project 410658).

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

90 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Cell growth inhibition

Remarks on result:

other: 59 to 136 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

165 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate reduction

Remarks on result:

other: 130 to 210 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

39 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: cell growth inhibition and growth rate

Details on results:

No inhibition of cell growth was observed at nominal test concentrations up to and including 80 mg/L. Algal cell growth was inhibited by 63% at the highest concentration tested, i.e. at 160 mg/L. No statistically significant inhibition of cell growth was found at nominal concentrations of 10 to 80 mg/L (Bonferroni t and Tukey test, = 0.05).
Growth rates were in the range of the blank-control at the concentrations from 10 to 80 mg/L during the 72 h test period, whereas the growth rate of algae exposed to 160 mg/L was significantly reduced, i.e. by 38%. No statistically significant reduction of growth rate was found at nominal concentrations of 10 to 80 mg/L (Bonferroni t and Tukey test, = 0.05).

Results of the analytical investigations:

Analyses showed that the measured concentrations at the start of the test were in agreement with nominal. Analysis was only considered for the nominal concentrations of 40, 80 and 160 mg/L, which were essential for determination of the toxicity parameters. Measured concentrations were 38 mg/L (96%), 73 mg/L (91%) and 149 mg/L (93%) at the expected nominal concentrations of 40, 80 and 160 mg/L, respectively. During the 72 h exposure period the measured concentrations decreased by more than 20% below initial. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated to correspond to 17, 39 and 114 mg/L, respectively.

RANGE FINDING TEST

Mean cell densities, inhibition of cell growth and reduction of growth rate

The results showed that the EC50 for both cell growth inhibition and growth rate reduction were above nominally 100 mg/L.

Stability of test substance under test conditions

The analytical results showed that measured test substance concentrations in samples taken from 10 and 100 mg/L decreased by more than 20% during the 72 h test period. The measured concentration at 100 mg/L decreased from 76 mg/L to 25 mg/L during the test period, while at 10 mg/L the measured concentration decreased from 8.6 to 0.44 mg/L during the test period.

FINAL TEST

Measured test substance concentrations

At the start of the test, the actual test concentrations were in agreement with nominal. Measured concentrations were 38 mg/L (96%), 73 mg/L (91%) and 149 mg/L (93%) at the expected nominal concentrations of 40, 80 and 160 mg/L. During the 72 h exposure period the measured

concentrations decreased by more than 20% below initial. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated.

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC50 for cell growth inhibition and growth rate (based on TWA concentrations) were determined to be 90 and 165 mg/L, respectively, while the NOEC for both the parameters was 39 mg/L.

Executive summary:

A study was conducted to assess the toxicity potential of the test substance to algae according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. In the range-finding test, exponentially growing algal cultures were exposed to test substance concentration range of 0.1 to 100 mg/L increasing by a factor of 10. In addition, a blank and treatment-control (with adjusted pH) were tested. The results showed that the EC50for both cell growth inhibition and growth rate reduction were above nominally 100 mg/L. The project was continued with a final test exposing exponentially growing algal cultures to a blank-control and nominal test substance concentrations of 10, 20, 40, 80 and 160 mg/L. In addition, a treatment-control with pH adjusted to that of the highest test substance concentration was included to correct for possible pH-related effects on algal cell growth at 160 mg/L. The initial algal cell density was 10^4 cells/mL. The total test period was 72 h. Samples for analyses were taken at the start, after 24 h of exposure and at the end of the 72 h test period.

Analyses showed that the measured concentrations at the start of the test were in agreement with nominal. Analysis was only considered for the nominal concentrations of 40, 80 and 160 mg/L, which were essential for determination of the toxicity parameters. Measured concentrations were 38 mg/L (96%), 73 mg/L (91%) and 149 mg/L (93%) at the expected nominal concentrations of 40, 80 and 160 mg/L, respectively. During the 72 h exposure period the measured concentrations decreased by more than 20% below initial. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated to correspond to 17, 39 and 114 mg/L, respectively. The study met the acceptability criteria prescribed by the protocol and was considered valid.

The 72 h EC50 for cell growth inhibition and growth rate based on TWA concentrations were determined to be 90 and 165 mg/L, respectively, while the NOEC for both the parameters was 39 mg/L (Migchielsen MHJ, 2004).

Description of key information

The 72 h EC50 for cell growth inhibition and growth rate based on TWA concentrations were determined to be 90 and 165 mg/L, respectively, while the NOEC for both the parameters was 39 mg/L.  Further, the 72 h EbC10 and ErC10 were determined to be at 44 and 49 mg/L respectively (TWA concentrations) respectively

Key value for chemical safety assessment

EC50 for freshwater algae:

165 mg/L

EC10 or NOEC for freshwater algae:

49 mg/L

Additional information

A study was conducted to assess the toxicity potential of the test substance to algae according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. In the range-finding test, exponentially growing algal cultures were exposed to test substance concentrations ranged from 0.1 to 100 mg/L increasing by a factor of 10. In addition, a blank and treatment-control (with adjusted pH) were tested. The results showed that the EC50 for both cell growth inhibition and growth rate reduction were above nominally 100 mg/L. The project was continued with a final test exposing exponentially growing algal cultures to a blank-control and nominal test substance concentrations of 10, 20, 40, 80 and 160 mg/L. In addition, a treatment-control with pH adjusted to that of the highest test substance concentration was included to correct for possible pH-related effects on algal cell growth at 160 mg/L. The initial algal cell density was 10⁴cells/mL. The total test duration was 72 h. Samples for analyses were taken at the start, after 24 h of exposure and at the end of the 72 h test period.

Analyses showed that the measured concentrations at the start of the test were in agreement with nominal. Analysis was only considered for the nominal concentrations of 40, 80 and 160 mg/L, which were essential for determination of the toxicity parameters. Measured concentrations were 38 mg/L (96%), 73 mg/L (91%) and 149 mg/L (93%) at the expected nominal concentrations of 40, 80 and 160 mg/L, respectively. During the 72 h exposure period the measured concentrations decreased by more than 20% below initial. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated to correspond to 17, 39 and 114 mg/L, respectively. The study met the acceptability criteria prescribed by the protocol and was considered valid.

The 72 h EC50 for cell growth inhibition and growth rate reduction based on TWA concentrations were determined to be 90 and 165 mg/L, respectively, while the NOEC for both the parameters was 39 mg/L. Further, the 72 h EbC10 and ErC10 were determined to be at 44 and 49 mg/L respectively (TWA concentrations) respectively (Migchielsen, 2004).