2-hydroxy-3-phenoxypropyl methacrylate

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:

August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-phenoxypropyl methacrylate

EC Number:

240-994-5

EC Name:

2-hydroxy-3-phenoxypropyl methacrylate

Cas Number:

16926-87-7

Molecular formula:

C13H16O4

IUPAC Name:

2-hydroxy-3-phenoxypropyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
171512
- Expiration date of the lot/batch: 23 December 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25ºC, below 70RH%) protected from humidity (tigh closed container)
- Solubility and stability of the test substance in the solvent/vehicle:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
A stock solution with a nominal concentration of 100.00 mg/L was prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) using ultrasonic bath (~30 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae

- Final dilution of a dissolved solid, stock liquid or gel:
100, 31.3, 9.8, 3.1, 1.0 mg/L

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Solution

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Analytical measurement was performed at the applied test concentration levels and from the control at the beginning and at the end of the experiment. The samples were analysed by HPLC system with UV detection method.

Test solutions

Vehicle:

yes

Remarks:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Details on test solutions:

A stock solution with a nominal concentration of 100.0 mg/L was prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) using ultrasonic bath (~ 30 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
- Method of cultivation: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4 and 22.7 °C measured in the flask and between 22.1 and 23.1 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.33 –8.80 during the experiment.

Nominal and measured concentrations:

Nominal: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L
Neasured: 0.99, 3.14, 9.90, 31.45 and 99.99 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mLErlenmeyer flasks
- Type: air-permeable stoppers
- Material, size, headspace, fill volume: 100mL algal suspension
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10^4 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
OTHER TEST CONDITIONS
- Adjustment of pH: NO
- Light intensity and quality: The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7594 lux (equivalent to ~103 μE/m2/s) , which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: 0.1, 1.0, 10.0, 100.0 mg/L
- Test concentrations: 0.99, 3.14, 9.90, 31.45 and 99.99 mg/L
- Results used to determine the conditions for the definitive study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.69 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

Effect conc.:

3.1 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:

LOEC

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 18/178-022AL) (before this test) with the reference item Potassium dichromate is (Batch Number: A0345704): 18 – 21 June 2018. The 72h ErC 50: 0.99 mg/L, (95 % confidence limits: 0.91 – 1.07 mg/L) The 72h EbC 50: 0.69 mg/L, (95 % confidence limits: 0.64 – 0.75 mg/L) The 72h EyC 50: 0.60 mg/L, (95 % confidence limits: 0.55 – 0.65 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Any other information on results incl. tables

Results Evaluation

Definitions:

Cell Density:        the number of cells per mL

Growth:                the increase of cell density over the test period

Biomass (b):         the actual number of cells per volume of medium (cells/mL) calculated as the area under the growth curve (A)

Yield (y):               the cell density at the end of the test minus the starting cell density

Average Specific

Growth Rate (m): the increase in cell density per time unit

E_bC₅₀:                  the calculated concentration of test item which results in a 50 % reduction of biomass (b) relative to the control

E_rC₅₀:                   the calculated concentration of test item which results in a 50 % reduction of growth rate (μ) relative to the control

E_yC₅₀:                   the calculated concentration of test item which results in a 50 % reduction of yield (y) relative to the control

NOEC:                 (No Observed Effect Concentration) the highest test concentration at which no significant inhibition of growth is observed relative to the control

LOEC:                  (Lowest Observed Effect Concentration) the lowest test concentration at which a significant inhibition of growth is observed relative to the control

 

Calculation of Average Specific Growth Rate

 

Concentration-effect relationship was calculated by comparing growth rates in control, test cultures in the following way.

The average specific growth rate (μ) for individual cultures are calculated from the following relationship:

 μ = (ln(N_n) - ln(N₀)) / (t_n - t₀)

 

Where        ln (N_n)  = natural logarithm of measured cell density at time t_n

      ln (N₀)  = natural logarithm of measured cell density at time t₀

      t₀          = time (hour) of the beginning of the test

      t_n          = time (hour) of n^thmeasurements after the beginning of the test

 

The percentage inhibition of growth rate (% Iµ):

 %Iµ = ((µ_c - µ_t) / µ_c) .100%

Where        % Iµ     = percent inhibition in average specific growth rate

                   µ_c        = mean growth rate of the control

                   µ_t       = mean growth rate of test concentration

 

Calculation of Area Under the Growth Curve

A = [(N₁ -N₀ / 2).t₁] + [((N₁ +N₂ -2N₀)/2).(t₂ -t₁)] + [((N_n-1 + N_n -2N₀)/2).(t_n-t_n-1)

Where         N₀        = nominalcell density at time t ₀ (start of the test)

 N₁        = mean measuredcell density at t₁ (24 hours)

 N₂        = mean measured cell density at t₂ (48 hours)

N_n        = mean measured cell density at t_n

t₁         = time of first measurement after start of the test

t₂         = time of second measurement after start of the test

t_n         = time of n^thmeasurement after start of the test

 The percentageinhibition of area (% I_A):

%I_A = ((A_c-A_t) / (A_c) . 100%

 

Where        % I_A     = percent inhibition in area under the growth curve

           A_c        = mean area of the control

           A_t        = mean area of test concentration

 

Calculation of Yield

 

Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated.

 

Percentage inhibition in yield (% Iv):

Iy = ((Yc-Yi)/(Yc)).100%

 

Where:       y_c =  mean value for yield in the control group
y_i =  mean value for yield for the test concentration

 

Area under the growth curve (biomass), average specific growth rate and yield were calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of PHPM test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates and areas were significantly different from that of the control group at the examined concentration range of 9.8 – 100.0 mg/L (nominal), therefore the NOEC was determined as 3.1 mg/L (nominal) and LOEC was determined as 9.8 mg/L (nominal). The 0-72 h yield was significantly different from that of the control group at the examined concentration range of 3.1 – 100.0 mg/L (nominal), therefore the NOEC was determined as 1.0 mg/L (nominal) and LOEC was determined as 3.1 mg/L (nominal). The biological results are summarised in the following table:

Table 8: Influence of PHPM on the Growth of Pseudokirchneriella subcapitata
Parameter Growth rate (r) Yeild (y) Biomass (b)
(0-72 h) [mg/L] [mg/L] [mg/L]
Calculation based on nominal test item
EC50 18.69 7.64 9.72
95% conf. limits 16.10-21.69 6.66-8.76 8.39-11.27
NOEC 3.1 1.0 3.1
LOEC 9.8 3.1 9.8

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic response was observed at the two highest examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 3.2) and one controlwere tested in the main experiment.

The nominal concentrations of test item used in the main experimentwere: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.

Test concentrations were analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test item concentrations were: 0.99, 3.14, 9.90, 31.45 and 99.99 mg/L.

As the analytically measured concentrations deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentrations.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software.

The E_rC₅₀, E_bC₅₀ and E_yC₅₀ values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates and areas were significantly different from that of the control group at the examined concentration range of 9.8 – 100.0 mg/L (nominal), therefore the NOEC was determined as 3.1 mg/L (nominal) and LOEC was determined as 9.8 mg/L (nominal). The 0-72 h yield was significantly different from that of the control group at the examined concentration range of 3.1 – 100.0 mg/L (nominal), therefore the NOEC was determined as 1.0 mg/L (nominal) and LOEC was determined as 3.1 mg/L (nominal). The biological results are summarised in the following table:

Table 1: Influence of PHPM on the Growth of Pseudokirchneriella subcapitata

Parameter (0 -72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on nominal test item concentrations
EC50 95% conf. limits	18.69  16.10 -21.69	7.64  6.66 -8.76	9.72  8.39 -11.27
NOEC LOEC	3.1 9.8	1.0 3.1	3.1 9.8