Phenol, mono- + distyrenated (MSP+DSP)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Novares LS 500; phenol, styrenated
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 28324
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
- Substance type: organic
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light

Analytical monitoring:

yes

Details on sampling:

Samples for TOC-analysis were taken at 0 h and 72 h: TOC-Analyser (DIMA-TOC 100).
Measurements of the cell concentrations per mL: at time 24 h, 48 h and 72 h by optical densitometry at 525 nm

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) prepared by stirring various amounts of the test item in Alga-medium
for 48 h with magnetic stirrers (open conditions). The WAFs were prepared in 2-L glass beakers.
The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loading rate of Novares LS 500 was weighed in a weighing scoop, that afterwards was placed into the glass beaker.
The beakers were then filled with Alga-medium.
Mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction
of the test item. To ensure this, the vortex developed at the surface by stirring was set at ~ 10 % of the water depth.
After stirring for 48 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation.
The extracts gained with this method were clear.

- Pre-conditioning: The test vessels of the Definite Alga-Test were rinsed with control media and WAFs for ca. 0.5 h before the definite
start of the tests to facilitate adsorption of the test item to the glass walls of the beakers.
Control media and WAFs were then discarded, and the test vessels were refilled again with control media and WAFs, respectively.
- Controls: without test material (blank; Alga medium)

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green alga
- Strain: : SAG 86.81
- Source: Albrecht von Haller Institut für Pflanzenwissenschaften der Universität Göttingen

- Age of inoculum (at test initiation): ca. 72 h
[Pre-culture: Incubation in 250 mL Erlenmeyer flasks at ± 25 °C on a shaker
(80 - 120 rpm), ca. 72 h, ca. 10000 lux (Gro-Lux fluorescent tubes), permanent illumination]

- Method of cultivation: Sterile on agar tubes (OECD-Medium, 0.8 % Agar), transferred to a new agar tube every 2 months

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no data specified: Compositions of stock solutions given.

Test temperature:

20.5- 21.5 °C

pH:

initial (with algae): 7.8 - 7.9
final (with algae): 8.4 (control); 8.8 (0.5 mg/L); 8.7 (1 mg/L); 8.6 (2.5 mg/L); 8.1 (5 mg/L); 8.0 (10 mg/L); 8.0 (25 mg/L)

Dissolved oxygen:

--

Salinity:

no data specified: Compositions of stock solutions given.

Nominal and measured concentrations:

Time [h] Control 0.5 mg/L 1 mg/L 2.5 mg/L 5 mg/l 10 mg/L 25 mg/L (loading)
==============================================================================
0 0.6 n.a. 1.2 n. a. 2.7 n.a. 5.6 mg C/L (TOC)
72 0.9 n.a. 1.0 n. a. 2.5 n.a. 5.5 mg C/L (TOC)
==============================================================================
n.a. not analyzed
Note: TOC values still include background.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL-Erlenmeyer
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: approx. 5000 cells/mL
- Control end cells density: approx. 452600 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted from deionised water and mineral stock solutions

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: permanent
- Light intensity and quality: approx. 10000 Lux (Gro-Lux fluorescent tubes)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: densitometrically at 525 nm (turbidity)
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 and 2.5
- Range finding study
- Results used to determine the conditions for the definitive study: based on results from daphnia test

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Remarks on result:

other: Loading was converted to a concentration value (0.36 mg TOC/L) based on measured concentrations of the test solutions. This value was corrected for the neat substance taking into account the carbon content of the substance

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Loading was converted to a concentration value (1.16 mg TOC/L) based on measured concentrations of the test solutions. This value was corrected for the neat substance taking into account the carbon content of the substance

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.14 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
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

- Observation of abnormalities (for algal test):
Observations by microscopy at the end of the testing period of 72 h:

Control: Appearance of alga cells was normal, cell proliferations could be observed regularly.
0.5 mg/L: Appearance of alga cells and cell proliferations comparable to control, however some of the alga cells were agglomerated.
1.0 mg/L: Comparable to 0.5 mg/L
2.5 mg/L: Appearance of alga cells comparable to control but lower number of cell proliferations.
5.0 mg/L: Alga cells were slightly deformed, low number of cells, cell proliferations could not be observed.
10 mg/L: Alga cells were deformed, low number of alga cells, cell proliferations could not be observed.
25 mg/L: Comparable to 10 mg/L

Reported statistics and error estimates:

standard deviations, coefficient of variance

Growth rates are listed below, from 0 -24, 24 - 48, and 48 - 72 h (Table 1) and average from 0 - 72 h (Table 2):

Table 1: Loading rates of the Test item, cells/mL in the test flasks at 24, 48 and 72 h with mean value,

section-by-section average specific growth rate [μ] during observation times 0-24 h, 24-48 h

and 48-72 h, mean value of the average specific growth rate [μ], standard deviation and

coefficient of variation Vc [%], inhibition [%] and mean value of inhibition of the average

specific growth rate [%] during observation times 0-24 h, 24-48 h and 48-72 h (Rep. Tab. 3)

[Note: Negative inhibition means increased growth, apparently stimulation]

Loading rate [mg/L]	Cells / mL	Cells / mL Mean value	Average Specific growth rate [µ]	Average Specific growth rate [µ] Mean value	Standard deviation	Coefficient of variation Vc [%]	Inhibition of Average Specific growth rate [%]	Inhibition of Average Specific growth rate Mean value [%]
	24 h
Control 1	12332	13394	0.91	0.98	0.13	13.49
Control 2	12332		0.91
Control 3	15518		1.14
0.5	12332	10236	0.91	0.71	0.17	23.92	7.79	27.75
0.5	9187		0.61				37.73
0.5	9187		0.61				37.73
1.0	12332	10236	0.91	0.71	0.17	23.92	7.79	27.75
1.0	9187		0.61				37.73
1.0	9187		0.61				37.73
2.5	6083	6083	0.20	0.20	0	0	79.65	79.65
2.5	6083		0.20				79.65
2.5	6083		0.20				79.65
5.0	3021	5063	-0.50	-0.03	0.40	1218	150.8	103.4
5.0	6083		0.20				79.65
5.0	6083		0.20				79.65
10	3021	3021	-0.50	-0.50	0	0	150.8	150.8
10	3021		-0.50				150.8
10	3021		-0.50				150.8
25	3021	3021	-0.50	-0.50	0	0	150.8	150.8
25	3021		-0.50				150.8
25	3021		-0.50				150.8
Loading rate [mg/L]	Cells / mL	Cells / mL Mean value	Average Specific growth rate [µ]	Average Specific growth rate [µ] Mean value	Standard deviation	Coefficient of variation Vc [%]	Inhibition of Average Specific growth rate [%]	Inhibition of Average Specific growth rate Mean value [%]
	48 h
Control 1	35503	41351	1.06	1.13	0.17	14.77
Control 2	46053		1.32
Control 3	42495		1.01
0.5	42495	46067	1.24	1.51	0.24	15.94	-9.73	-34.12
0.5	46053		1.61				-42.98
0.5	49653		1.69				-49.65
1.0	38979	41337	1.15	1.40	0.23	16.65	-2.07	-24.41
1.0	46053		1.61				-42.98
1.0	38979		1.45				-28.18
2.5	32069	30938	1.66	1.63	0.06	3.97	-47.44	-44.13
2.5	32069		1.66				-47.44
2.5	28677		1.55				-37.52
5.0	3021	4042	0.00	-0.23	0.40	173.2	100.0	120.7
5.0	6083		0.00				100.0
5.0	3021		-0.70				162.1
10	3021	3021	0.00	0	0	- *	100.0	100.0
10	3021		0.00				100.0
10	3021		0.00				100.0
25	3021	3021	0.00	0	0	- *!	100.0	100.0
25	3021		0.00				100.0
25	3021		0.00				100.0
Loading rate [mg/L]	Cells / mL	Cells / mL Mean value	Average Specific growth rate [µ]	Average Specific growth rate [µ] Mean value	Standard deviation	Coefficient of variation Vc [%]	Inhibition of Average Specific growth rate [%]	Inhibition of Average Specific growth rate Mean value [%]
	72 h
Control 1	139285	153153	1.37	1.31	0.07	5.49
Control 2	157730		1.23
Control 3	162444		1.34
0.5	176835	178544	1.43	1.36	0.12	9.01	-8.59	-3.21
0.5	191597		1.43				-8.58
0.5	167200		1.21				7.53
1.0	157730	170480	1.40	1.42	0.06	4.14	-6.47	-8.02
1.0	181714		1.37				-4.54
1.0	171997		1.48				-13.06
2.5	76004	67330	0.86	0.75	0.24	32.08	34.28	42.88
2.5	79934		0.91				30.44
2.5	46053		0.47				63.92
5.0	6083	5063	0.70	0.23	0.81	346.4	46.69	82.23
5.0	3021		-0.70				153.3
5.0	6083		0.70				46.69
10	3021	3021	0.00	0	0	- *	100.0	100.0
10	3021		0.00				100.0
10	3021		0.00				100.0
25	3021	3021	0.00	0	0	- *!	100.0	100.0
25	3021		0.00				100.0
25	3021		0.00				100.0

Table 2: Loading rate of the Test item, cells/mL in the test flasks at 72 h, average specific growth

rate [μ] over the whole testing period 0-72 h, mean value of the average specific growth

rate [μ], standard deviation and coefficient of variation Vc [%], inhibition [%] and mean value

of inhibition [%] of the average specific growth rate [μ] (Rep. Tab. 4)

Loading rate [mg/L]	Cells / mL	Average Specific growth rate [µ]	Average Specific growth rate [µ] Mean value	Standard deviation	Coefficient of variation Vc [%]	Inhibition of Average Specific growth rate [%]	Inhibition of Average Specific growth rate Mean value [%]
	72 h
Control 1	139285	1.11	1.14	0.03	2.38
Control 2	157730	1.15
Control 3	162444	1.16
0.5	176835	1.19	1.19	0.02	1.91	-4.26	-4.50
0.5	191597	1.22				-6.61
0.5	167200	1.17				-2.63
1.0	157730	1.15	1.18	0.02	2.02	-0.92	-3.15
1.0	181714	1.20				-5.06
1.0	171997	1.18				-3.45
2.5	76004	0.91	0.86	0.10	11.84	20.40	24.79
2.5	79934	0.93				18.93
2.5	46053	0.74				35.03
5.0	6083	0.07	-0.01	0.13	1218	94.15	101.0
5.0	3021	-0.17				114.6
5.0	6083	0.07				94.15
10	3021	-0.17	-0.17	0	0	114.6	114.6
10	3021	-0.17				114.6
10	3021	-0.17				114.6
25	3021	-0.17	-0.17	0	0	114.6	114.6
25	3021	-0.17				114.6
25	3021	-0.17				114.6

Validity criteria fulfilled:

yes

Remarks:

1) Biomass in controls increased 31x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 18.1 % (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.4 % (nom. value ≤ 7%).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given; guideline study (OECD TG 201); test was performed by a laboratory with a high reputation for delivery of solid data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Phenol, styrenated; Mono (or di or tri)-(alpha-methylbenzyl) phenol
- no further information

Analytical monitoring:

yes

Details on sampling:

- no data reported

Vehicle:

not specified

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- no data reported

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Nominal and measured concentrations:

- graduate doses of test substance were applied. Individual concentrations are not reported.

Details on test conditions:

- no data reported

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Results are reported without any detail in databases (CHRIP/J-CHECK) of the National Institute of Technology and Evaluation (NITE) of Japan.

Validity criteria fulfilled:

not specified

Conclusions:

In a test according to OECD TG 201, the 72 h EC50 and NOEC of phenol, styrenated to algae were determined to be >10 and 3.2 mg/L respectively.

Description of key information

In a 72 h growth inhibition test to algae (GLP study according to OECD TG 201, an EL50 and a NOELR (nominal load of water-accomodated fractions) have been determined. Using measured concentrations of the WAFs, an EC50 of 1.35 mg/L and a NOEC of 0.42 mg/L are obtained.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.35 mg/L

EC10 or NOEC for freshwater algae:

0.42 mg/L

Additional information

For the substances phenol, styrenated and phenol, mono- & distyrenated, two growth inhibition studies with algae have been identified.

One is a GLP study according to OECD TG 201 using Novares LS 500 as test substance (Aniol/STZ 2010). Reliability is 1. The study is used as key study.

The second test was carried out for the National Institute of Technology and Evaluation/Ministry of the Environment of Japan and is reported in the Japanese databases CHRIP/J-CHECK (CHRIP/NITE 1997). The study was performed according to OECD TG 201, but reporting is very limited. Details on test performance and results are not available from the database. Test substance was phenol, styrenated (CAS 61788-44-1). Composition of the test substance is not reported, but from the substance name of the regulatory program (mono (or di or tri)-(alpha-methylbenzyl)phenol) it can be concluded that the test substance is a mixture of mono-, di-, and tristyrenated phenol. Similarity with Novares LS 500 is considered to be adequate to use data from this test as weight of evidence for the characterisation of Novares LS 500.

 

Aniol/STZ 2010

Due to the limited solubility and multi-constituent composition of the test substance, the test was performed using water-accommodated fractions. A 72h EL50 of 3.14 mg/L was determined (nominal loading). The 72h NOELR (no observed effect loading rate) was 1 mg/L.

Dissolved concentrations of controls and of selected test solutions were measured at the beginning and at the end of the test period. TOC was analysed as representative parameter for the test substance. As nominal loads differ considerably from measured concentrations, the substance concentration representing 50% (EC50) and no (NOEC) growth inhibition is derived from the respective nominal loadings. For determination of concentrations, blank corrected geometric means are used.

 

Loadings (mg substance/L)		control	1 mg/L	5 mg/L	25 mg/L
measured concentrations (mg TOC/L) at	0 h	0.6	1.2	2.7	5.6
	72 h	0.9	1.0	2.5	5.5
geometric mean (mg TOC/L)		0.735	1.095	2.598	5.55
corrected conc. (mg TOC/L)			0.36	1.86	4.82

 

From the blank corrected measured concentrations (mg TOC/L), an EC50 was derived by linear interpolation between NOEL and EL50. Using the mean carbon content of the substance (86.018 %) determined by carbon content and percentage of the individual components, the respective substance concentrations for NOEC and EC 50 were calculated.

 

	Loading (mg substance/L)	Concentration (mg TOC/L)	Concentration (mg substance/L)
E(L)C50	3.14	1.16	1.35
NOE(L)C	1	0.36	0.42

 

CHRIP/NITE 1997

In this algal growth inhibition test, EC50 and NOEC were determined. Effect concentration is based on growth rate.

72 h EC50:        > 10 mg/L

72 h NOEC:        3.2 mg/L

 

The lower results (Aniol/STZ 2010) are taken for further consideration.

 

In addition as supporting evidence, read across from the Substance LA 700 (phenol, methylstyrenated) was used. Transformation of loads to measured concentrations results in an EC50 in the same order of magnitude as for LS 500 (phenol, mono- & distyrenated).