4-phenylbenzophenone

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:

25 August to 03 October 2017 (experimental period)

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:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

DETAILS ON TEST MATERIAL
- Source and lbatch: IGM Resins, batch no. 20161118
- Expiration date of the lot/batch: 17/11/2017
- Purity test date: 16/11/2016
- Purity: 99.74%
- Test facility test item number: 208643/A
- Solubility in water: 0.0736 mg/L (determined at Charles River Laboratores Den Bosch).
- Appearance: white to off-white crystalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: no data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations
- Sampling method: 2.0 mL at 0, 24 and 72 hours. At 72 hours replicates with algae were pooled before sampling at 10, 18, 32 and 56% of the saturated solution. At 100 mg/L (100% saturated solution) replicates 2 and 3 were pooled before sampling.
- Sample storage conditions before analysis: Stored in a freezer (

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solution with a nominal loading rate of 100 mg/L were prepared by dissolving the test substance in algal culture medium. This solution was stirred for 3 days with magnetic stirrer to achive maximum dissolution of the test substance in the test medium. The aqueous mixture was filtered through 0.45 um membrane filter (RC55, Whatman). The resultant saturated solution was used as the highest test concentration. Lower test concentrations were prepared by dilution of aliquots of the saturated solution in algal test medium.
- Eluate: M2 (according to OECD 201 guideline) formulated with Milli-RO water
- Differential loading: No
- Controls: Algal test medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Test solutions were observed to be clear and colourless at the end of the preparation procedure

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days

ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21.0 - 23.5oC

pH:

7.9 - 8.1

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L
Measured concentrations: 6.6, 12, 23, 26, 34 and 40 ug/L (based on time weighted average)

Details on test conditions:

CULTURE OF ALGAE
-Stock culture: 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, with a light intensity of 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Growth medium used: M1; 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).
- Composition: NaNO3 500 mg/L, K2HPO4.3H2O 52mg/L, MgSO4.7H2O 75mg/L, NaCO3.10H2O 54 mg/L, C6.H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7xH2O 6 mg/L, H3BO4 2.9 mg/L.

PRE-CULTURE OF ALGAE FOR TEST
- 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.
-Growth medium: M2 according to the OECD 201 Guideline, formulated using Milli-RO water.
- Composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 mg/L, NaEDTA.2H2O 100 mg/L, H3BO3 185 mg/L, MnCl2.4H2O 415 mg/L, ZnCl2 3 mg/L, CoCl2.6H2O 1.5 mg/L, CuCl2.2H2O 0.01 mg/L, NaMoO4.2H2O 7 mg/L.

TEST SYSTEM
- Test vessel: not stated
- Type (delete if not applicable): closed (capped)
- Test vessels: 100ml, glass vessels, containing 50ml of test solution.
- Aeration: continuous shaking.
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 1x104 cells/ml
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (1 or 2 replicates of each test concentrations without algae, 1 or 2 replicates of each test concentrations and the control for sampling after 24 hours)
- Test medium: M2 as detailed above.

OTHER TEST CONDITIONS
- Sterile test conditions: not stated
- Adjustment of pH: No
- Photoperiod: Continous illumination
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 97 to 98 µE.m-2.s-1.
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 10 mm). Algal medium was used as blank.

TEST CONCENTRATIONS
- Range finding study: a range finder study/combined limit study was conducted to determine the testing concentrations.
- Test concentrations: range finder was conducted at 1.0 and 10% saturation solution concentrations.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (further details provided below)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Biological relevance

Effect conc.:

12 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Statistical significance

Effect conc.:

< 6.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks:

(yield)

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Biological relevance

Effect conc.:

< 6.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks:

(yield)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 6.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks:

(yield)

Details on results:

effect was statistically significant but biologically not relevant (<10%)

Results with reference substance (positive control):

The Ec50 for growth rate inhibition, 72 hour ECr50 was 1.1mg/L (95% confidence limits of 1.1 to 1.1 mg/L). This lies within the historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L.
The EC50 for yield inhibition (72 hour EyC50) was 0.36mg/L with a 95% confidence limit from 0.35 to 0.36mg/L. The historical range lies between 0.43 and 1.1mg/L.

Effect parameters	Results based on time-weighted average measured concentrations (ug/L)
	NOEC (statistical significance)	NOEC (biological relevance)	EC10	EC20	EC50
Growth rate	<6.6	12	33 (30-35)	35 (33-38)	41 (37-45)
Yield	<6.6	<6.6	14 (9.5-19)	17 (12-24)	26 (17-39)

Values in parenthesis are 95% confidence limit

Growth rate and percentage inhibition at different time intervals.

4-PBZ concentration TWA (µg/L)	N	0-24 hours		24-48 hours		48-72 hours
		Mean	% Inhibition	Mean	% inhibition	Mean	% inhibition
Control	6	2.145		2.003		1.607
6.6	3	2.325	-8.4	1.622	19.0	1.607	0.0
12	3	2.303	-7.4	1.756	12.3	1.519	5.5
23	3	2.129	0.7	1.547	22.8	1.512	5.9
34	3	1.805	15.8	1.369	31.7	1.581	1.7
40	3	0.639	70.2	1.083	45.9	1.359	15.5

Yield and percentahe inhibition for the total test period.

4-PBZ concentration TWA (µg/L)	Mean	Standard deviation	N	% inhibition
Control	315.3	15.93	6
6.6	257.2	5.53	3	18.4*
12	264.2	18.09	3	16.2*
23	178.7	17.39	3	43.3*
34	115.4	9.50	3	63.4*
40	24.1	16.80	3	92.4*

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour NOEC for growth rate inhibition for green algae (Pseudokirchneriella subcapitata) was 12 ug/L based on biological relevance and the EC50 was 41 ug/L based on time weighted mean concentrations.

Executive summary:

A 72-hour toxicity study with the green alga, Pseudokirchneriella subcapitata, was conducted in accordance with the OECD test guideine 201 and in compliance with GLP.

The test was performed at nominal concentrations of 10, 18, 32, 56 and 100 mg/L, with test solutions prepared by dilution from a 100 mg/L saturated solution. The time-weighted measured concentrations were 6.6, 12, 23, 26, 34 and 40 ug/L, respectively.

Under the conditions of the test the EC50 for growth rate inhibition was 41 ug/L (37-45 ug/L 95% CI). The NOEC for growth rate inhibition was 12 ug/L, based on biological relevance.

This toxicity study is considered acceptable and satisfies the guideline requirement for toxicity to aquatic algae.

Description of key information

A 72-hour toxicity study with the green alga, Pseudokirchneriella subcapitata, was conducted in accordance with the OECD test guideline 201 and in compliance with GLP.

The test was performed at nominal concentrations of 10, 18, 32, 56 and 100 mg/L, with test solutions prepared by dilution from a 100 mg/L saturated solution. The time-weighted measured concentrations were 6.6, 12, 23, 26, 34 and 40 ug/L, respectively.

Under the conditions of the test the EC50 for growth rate inhibition was 41 ug/L (37-45 ug/L 95% CI). The NOEC for growth rate inhibition was 12 ug/L, based on biological relevance.

This toxicity study is considered acceptable and satisfies the guideline requirement for toxicity to aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

41 µg/L