Distillates (coal tar), benzole fraction

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:

12 Aug. - 12 Nov. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Distillates (coal tar), benzole fraction

EC Number:

283-482-7

EC Name:

Distillates (coal tar), benzole fraction

Cas Number:

84650-02-2

Molecular formula:

not applicable

IUPAC Name:

Distillates (coal tar), benzole fraction

Details on test material:

- Name of test material (as cited in study report): light oil
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: liquid
- Stability under test conditions: WAF (water accommodated fraction) limited
- Storage condition of test material: room temperature, exclusion of light

Sampling and analysis

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

Test solutions

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 24 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 rates of Light Oil were put directly into the beakers. 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 24 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.
- Differential loading: control, 10, 25 mg/L, 50 mg/L, 100 mg/L, 250 mg/L, and 500 mg/L
- Controls: without test material (blank; Alga medium)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

no data specified: Compositions of stock solutions given.

Test temperature:

23.9 - 24.7 °C (25 +-2°C)

pH:

initial (with algae): 7.8
final (with algae): 10.2 (control); 8.6 (10 mg/L); 8.1 (25 mg/L); 8.1 (50 mg/L); 7.9 (100 mg/L); 8.1 (250 mg/L); 7.9 (500 mg/L)

Salinity:

no data specified: Compositions of stock solutions given.

Nominal and measured concentrations:

Time [h] Control 10 mg/L 100 mg/L 500 mg/L loading
=====================================================
0 1.0 1.0 1.9 7.1 mgC/mL (TOC of WAFs)
72 0.6 1.1 2.5 8.1 mgC/mL (TOC of WAFs)
=====================================================

The oil-related TOC was about 0 mg C/L for 10 mg oil/L, 1.0 mg C/L for 100 mg/L, and 7.0 mg C/L for 500 mg oil/L.

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. 341500 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: deionised


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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

>= 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass (cell yield)

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): 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.
10 mg/L: Comparable to control, lower number of cells.
25 mg/L: Comparable to 10 mg/L, only a few cell proliferations could be observed.
50 mg/L: Alga cells were slightly deformed, cell proliferations could not be observed.
100 mg/L: Comparable to 50 mgL
250 mg/L: Comparable to 50 mgL, lower number of cells.
500 mg/L: Comparable to 50 mgL, very low number of cells.

Reported statistics and error estimates:

No data: due to high fluctuation of cell number in single replicate tests at the low concentrations statistics were not considered reasonable and reliable.

Any other information on results incl. tables

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	18746	19836	1.32	1.38	0.09	6.75
Control 2	22015		1.48
Control 3	18746		1.32
10	15518	14456	1.13	1.06	0.13	12.6	17.6	13.2
10	15518		1.13				17.6
10	12332		0.90				34.4
25	15518	14456	1.13	1.06	0.13	12.6	17.6	13.2
25	12332		0.90				34.4
25	15518		1.13				17.6
50	9187	9187	0.61	0.61	0.00	0.00	55.8	55.8
50	9187		0.61				55.8
50	9187		0.61				55.8
100	12332	11284	0.90	0.80	0.17	21.1	34.4	41.5
100	9187		0.61				55.8
100	12332		0.90				34.4
250	3021	5063	0.00 *	0.13	0.12	86.6	100	90.5
250	6083		0.20				85.7
250	6083		0.20				85.7
500	6083	5063	0.20	0.13	0.12	86.6	85.7	90.5
500	6083		0.20				85.7
500	3021		0.00 *				100
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	76004	77396	1.40	1.36	0.06	4.28
Control 2	87917		1.38
Control 3	68269		1.29
10	38979	36717	0.92	0.92	0.08	8.85	32.2	32.0
10	42495		1.01				25.9
10	28677		0.84				37.9
25	28677	29808	0.61	0.73	0.11	15.8	54.8	46.4
25	28677		0.84				37.9
25	32069		0.73				46.6
50	25325	24222	1.01	0.97	0.08	8.36	25.4	28.8
50	25325		1.01				25.4
50	22015		0.87				35.7
100	18746	19836	0.42	0.57	0.15	25.8	69.2	58.0
100	18746		0.71				47.5
100	22015		0.58				57.4
250	6083	4042	0.70	0.23	0.40	173	48.5	82.8
250	3021		0.00 **				100
250	3021		0.00 **				100
500	3021	3021	0.00 **	0.00	000	-	100	100
500	3021		0.00 **				100
500	3021		0.00				100
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	323078	341557	1.45	1.49	0.06	3.94
Control 2	378516		1.46
Control 3	323078		1.55
10	96065	103097	0.90	1.04	0.16	14.9	39.4	30.0
10	117159		1.01				31.8
10	96065		1.21				18.7
25	35503	36662	0.21	0.21	0.01	5.13	85.6	86.1
25	35503		0.21				85.6
25	38979		0.20				86.9
50	42495	38993	0.52	0.48	0.04	9.09	65.2	68.0
50	38979		0.43				71.0
50	35503		0.48				67.9
100	38979	41337	0.73	0.73	0.00	0.48	50.8	50.6
100	38979		0.73				50.8
100	46053		0.74				50.4
250	28677	23146	1.55	1.79	0.22	12.3	-4.26	-20.2
250	22015		1.99				-33.6
250	18746		1.83				-22.7
500	3021	3021	0.00	0.00	0.00	-	100	100
500	3021		0.00				100
500	3021		0.00				100

* The measurements of the cell number by optical densitometry at 525 nmgave values quite below the initial cell concentration of 5000 cells/mL.

Because of the uncertainty of the measurements in this lower absorbance range, the average specific growth rate [µ] has been assigned zero.

** The number of cells/mL was lower at observation time 72 h compared to 48 h. This would lead to a negative value of the average specific growth rate [µ],

which therefore has been assigned zero.

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	323078	1.39	1.41	0.03	2.17
Control 2	378516	1.44
Control 3	323078	1.39
10	96065	0.99	1.01	0.04	3.79	30.0	28.4
10	117159	1.05				25.3
10	96065	0.99				30.0
25	35503	0.65	0.66	0.02	2.71	53.6	52.8
25	35503	0.65				53.6
25	38979	0.68				51.4
50	42495	0.71	0.68	0.03	4.38	49.3	51.4
50	38979	0.68				51.4
50	35503	0.65				53.6
100	38979	0.68	0.70	0.03	4.57	51.4	50.0
100	38979	0.68				51.4
100	46053	0.74				47.4
250	28677	0.58	0.51	0.07	14.2	58.6	64.1
250	22015	0.49				64.9
250	18746	0.44				68.7
500	3021	0.00 *	0.00	0.00	-	100	100
500	3021	0.00 *				100
500	3021	0.00 *				100

* The measurements of the cell number by optical densitometry at 525 nmgave values quite below the initial cell concentration of 5000 cells/mL.

Because of the uncertainty of the measurements in this lower absorbance range, the average specific growth rate [µ] has been assigned zero.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1) Biomass in controls increased 68x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 6.1% (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.2% (nom. value ≤ 7%).