3,5-xylenol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Principles of method if other than guideline:

Deviations: The temperature in the test solutions deviated from 24±1°C and was instead in the range 22 - 23°C between teh 48 and 72 hour observaton periods. This was not considered to adversely impact the integrity of the study.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Mixed xylenols

IUPAC Name:

Mixed xylenols

Details on test material:

- Name of test material (as cited in study report):
- Substance type: phenol
- Physical state: not stated
- Analytical purity: 99.74%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition: 2,5-xylenol: 16.4%; 3,4-xylenol: 16.9%; 2,4-xylenol: 22.7%; 3,5-xylenol: 11.1%; 2,3-xylenol: 18.2%; 2,6-xylenol: 14.7%;
- Purity test date: not stated
- Lot/batch No.: 20NOV2003
- Expiration date of the lot/batch: 31 July 2005
- Stability under test conditions: not stated
- Storage condition of test material: Room temperature in the original container in a dark ventilated cabinet

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 1648
- Source (laboratory, culture collection): Universtiy of Texas, Austin, Texas, USA
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Continuous agitation on an orbital shaker (100 rpm) in 125 mL glass flasks each containing 50 mL of medium, covered with stainless steel caps permitting gas exchange.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, except screw cap tops used during test.
- Any deformed or abnormal cells observed: none stated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22-24°C

pH:

pH 8.2-8.4 (test start), pH 8.9-9.5 (test end)

Nominal and measured concentrations:

Nominal: 1.6, 3.1, 6.3, 13 and 25 mg/L
Measured: 1.0, 1.7, 4.4, 10 and 22 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass Erlenmeyer flasks with screw cap tops, 100 mL per flask.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): static test
- Renewal rate of test solution (frequency/flow rate): closed system
- Initial cells density: approx. 1.0 x 10E4 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile de-ionised water
- Total organic carbon: 1.7 mg/L
- Particulate matter: not observed
- Metals: not present in toxic concentrations
- Pesticides: not present in toxic concentrations
- Chlorine: not stated
- Alkalinity: not stated
- Ca/mg ratio: not stated
- Conductivity: 350 - 380 µmhos/cm
- Culture medium different from test medium:
- Intervals of water quality measurement: 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7000 - 8600 lux, photosynthetically active radiation (PAR) was 95 - 127 µE/m2/s
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: not stated

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: control, solvent control, 0.010, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 12-15 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test):
- Unusual cell shape: In the 10 mg/L exposure solution, cells were observed to be bloated at 48 hours. In the 10 and 22 mg/L exposure solutions, at 72 hours cells were observed to be bloated and cell fragments were seen.
- Colour differences: not stated
- Flocculation: not stated
- Adherence to test vessels: not stated
- Aggregation of algal cells: not stated
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no

Any other information on results incl. tables

Analytical analysis of the test media showed the mean measured exposure concentration ranged between 53 - 93% of nominal, i.e. 1.0, 1.7, 4.4, 10 and 22 mg/L. At

the end of the exposure period, the analytical result from the 6.3 mg/L nominal dose without algae present was 4.6 mg/L. From the 6.3 mg/L test solution with algae present, the measured concentration was 3.3 mg/L. This demonstrates that the presence of algae had a slight impact on the concentration of mixed xylenols in the test solution. Percentage recoveries in teh six QC samples demonstrated consistency with teh predetermined recovery range (60.4 - 65.2% for the 1.00 mg/L samples and 87.4 - 100% for the 10.0 and 50.0 mg/L samples).

Cell densities were determined at each observation interval. In the 10 mg/L exposure solution, cells were observed to be bloated at 48 hours. In the 10 and 22 mg/L exposure solutions, at 72 hours cells were observed to be bloated and cell fragments were seen. Cells exposed to 1.0, 1.7 and 4.4 mg/L were observed to be normal. The 72 hour cell density in the control and solvent control had a mean of 32.67 and 83.25 x 10E4 cells/mL, respectively. Cell densities in the 1.0, 1.7, 4.4, 10 and 22 mg/L treatment levels had a mean of 82.67, 83.00, 72.1, 53.00 and 13.92 x 10E4 cells/mL, respectively. A significant difference was observed in the total biomass between the control and solvent control (t-test) and so solvent control data were used to determine the treatment level effects. A significant reduction in total biomass was observed in the 4.4, 10 and 22 mg/L treatment levels compared to the solvent control data, using Williams' test.

Mean growth rates were 1.17 and 1.49 daysE-1 for teh control and solvent control, respectively after 72 hours. The t-test determined a significant difference between teh control and solvent control growth rate and so solvent control data were used to determine treatment related effects. The mean growth rates in the 1.0, 1.7, 4.4, 10 and 22 mg/L treatments were 1.49, 1.49, 1.44, 1.34 and 0.89 daysE-1, respectively. These data showed a significant reduction in growth rate with Williams' test for teh 4.4, 10 and 22 mg/L treatment levels, compared to the solvent control data.

Table 1: Concentrations of mixed xylenols measured during the 72 hour exposure of Pseudokirchneriella subcapitata

Nominal concentration (mg/L)	Measured concentration (mg/L)
	0 hour	72 hours	Mean	% of nominal
Control	<0.14	<0.14	NA	NA
Solvent control	<0.14	<0.14	NA	NA
1.6	1.3	0.70	1.0	63
3.1	2.2	1.1	1.7	53
6.3	5.5	3.3/4.6	4.4	69
13	11	9.4	10	79
25	24	21	22	90

 

Table 2: Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to mixed xylenols

Mean measured concentration (mg/L)	Cell density (x 10^4cells/mL)
	Observation interval (hours)
		24	48	72
Control	A	9.50	35.75	33.50
	B	3.50	26.50	35.25
	C	4.00	20.25	29.25
	Mean (SD)	5.67 (3.33)	27.50 (7.8)	32.67 (3.09)
Solvent control	A	2.75	25.25	85.00
	B	6.25	27.25	84.25
	C	5.25	34.75	80.50
	Mean (SD)	4.75 (1.8)	29.08 (5.01)	83.25 (2.41)
1.0	A	6.00	26.50	75.75
	B	6.00	32.75	81.25
	C	6.00	19.75	91.00
	Mean (SD)	6.00 (0)	26.33 (6.5)	82.67 (7.72)
1.7	A	4.25	28.25	84.00
	B	4.25	32.25	85.75
	C	6.25	28.25	79.25
	Mean (SD)	4.92 (1.15)	29.58 (2.31)	83.00 (3.36)
4.4	A	3.75	25.50	70.00
	B	4.50	21.50	78.25
	C	6.25	21.25	68.25
	Mean (SD)	4.83 (1.28)	22.75 (2.38)	72.17 (5.34)
10	A	3.50	15.00	51.50
	B	6.25	18.75	57.75
	C	4.75	17.75	49.75
	Mean (SD)	4.83 (1.38)	17.17 (1.94)	53.00 (4.21)^ab
22	A	1.25	8.00	15.75
	B	1.50	8.50	12.25
	C	3.00	6.75	13.75
	Mean (SD)	1.92 (0.95)	7.75 (0.9)^a	13.92 (1.76)^ab

SD = Standard deviation.
^a= Cells were observed to be bloated.
^b= Cell fragments were observed.

 

Table 3: Calculated biomass (area under the growth curve) of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to mixed xylenols

Mean measured concentration (mg/L)	Biomass (x 10^4cells.days/mL)
	Observation interval (hours)
		0-24	24-48	48-72	72 hour total area	% inhibition^a
Control	A	4.52	23.20	28.02	55.74	-
	B	1.33	15.02	24.90	41.24	-
	C	1.59	11.94	19.79	33.32	-
	Mean (SD)	2.48 (1.77)	16.72 (5.82)	24.24 (4.15)	43.43 (11.37)	NA
Solvent control	A	0.93	13.95	45.10	59.98	-
	B	2.79	16.90	45.62	65.31	-
	C	2.26	20.39	47.19	69.83	-
	Mean (SD)	1.99 (0.96)	17.08 (3.22)	45.97 (1.08)	65.04 (4.93)	NA
1.0	A	2.66	16.36	41.77	60.79	-
	B	2.66	19.71	46.67	69.04	-
	C	2.66	12.74	45.31	60.71	-
	Mean (SD)	2.66 (0)	16.27 (3.49)	44.58 (2.53)	63.51 (4.79)	2
1.7	A	1.73	16.36	45.94	64.03	-
	B	1.73	18.51	48.33	68.57	-
	C	2.79	17.43	43.96	64.18	-
	Mean (SD)	2.08 (0.61)	17.43 (1.07)	46.08 (2.19)	65.59 (2.58)	-1
4.4	A	1.46	14.62	38.96	55.04	-
	B	1.86	12.88	40.73	55.46	-
	C	2.79	13.68	36.46	52.93	-
	Mean (SD)	2.04 (0.68)	13.72 (0.87)	38.72 (2.15)	54.48 (1.36)^b	16
10	A	1.33	8.85	26.87	37.05	-
	B	2.79	12.34	31.04	46.17	-
	C	1.99	11.00	27.29	40.28	-
	Mean (SD)	2.04 (0.73)	10.73 (1.76)	28.40 (2.29)	41.17 (4.62)	37
22	A	0.13	3.89	9.06	13.08	-
	B	0.27	4.29	7.81	12.37	-
	C	1.06	4.16	7.71	12.93	-
	Mean (SD)	0.49 (0.50)	4.11 (0.20)	8.19 (0.75)	12.79 (0.38)^b	80

SD = Standard deviation
NA = Not applicable
^a= % inhibition relative to the solvent control.
^b= Significantly reduced compared to the solvent control, based on Williams’ test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 for total biomass was determined to be 14 mg/L and the NOEC was 1.7 mg/L.
The 72 hour EC50 for growth rate was determined to be >22 mg/L and the NOEC was 1.7 mg/L.