Tar bases, coal, crude

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

04 Oct. – 12 Nov. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline 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

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Coal tar bases
- 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: Test solutions were prepared in a dilution series of a stock solution of 100 mg/L test item in Alga-medium.
The stock solution and each degree of dilution were emulsified at 13500 rpm for 5 minutes using a mixing device
(Ultra-Turrax, Janke & Kunkel).
The test solutions were slightly turbid at test concentrations of 50, 25 and 10 mg/L.
Turbidity could not be observed at test concentrations ≤5 mg/L.

- Test concentrations: Control, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L, 25 mg/L, 50 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): slight turbidity at 50, 25 and 10 mg/L, but no precipitation.

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:

23.8- 24.7 °C (25 +-2°C)

pH:

initial (with algae): 7.9
final (with algae): 9.5 (control); 10 (1 mg/L); 8.9 (2.5 mg/L); 8.3 (5 mg/L); 8.3 - 8.1 (10 - 50 mg/L)

Dissolved oxygen:

not measured

Salinity:

no data specified: Compositions of stock solutions given.

Nominal and measured concentrations:

Time [h] Control 1 mg/L 10 mg/L 50 mg/L nominal concentration
========================================================
0 0.5 1.4 7.0 34.5 mg C/mL (TOC of WAFs)
72 1.6 1.6 6.8 34.1 mg C/mL (TOC of WAFs)
=====================================================
The tar-base dependent TOC was about <1 mg C/L for 1 mg oil/L, 5.5 - 6.5 mg C/L for 10 mg oil/L and 33 - 34 mg C/L for 50 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. 150,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3



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: by microscopy with counting chambers at time 24 h, 48 h and 72 h.
- 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

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass (cell yield)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass (cell yield)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 50 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: at 24 - 48 h at 5 an 10 mg/L, slightly at 48 - 72 h at 2.5 mg/L
- 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.
1 mg/L: Comparable to control.
2.5 mg/L: Comparable to control.
5 mg/L: Comparable to control.
10 mg/L: Appearance of alga cells was normal, only a few cell proliferations could be observed.
25 mg/L: Appearance of alga cells was normal, only a few cell proliferations could be observed.
50 mg/L: Agglomerates of alga cells, cell proliferations could not be observed.

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]

Concentration [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	15500	13833	1.13	1.01	0.13	12.7
Control 2	14000		1.03
Control 3	12000		0.88
1	14500	13667	1.06	1.00	0.06	5.52	-5.19	0.76
1	13500		0.99				1.87
1	13000		0.96				5.60
2.5	14000	12000	1.03	0.87	0.17	19.5	-1.72	14.4
2.5	10000		0.69				31.5
2.5	12000		0.88				13.5
5	12000	12000	0.88	0.87	0.13	14.5	13.5	14.0
5	10500		0.74				26.7
5	13500		0.99				1.87
10	16500	12500	1.19	0.89	0.30	34.2	-18.0	12.5
10	12000		0.88				13.5
10	9000		0.59				41.9
25	13500	14000	0.99	1.01	0.23	23.0	1.87	0.066
25	11000		0.79				22.1
25	17500		1.25				-23.8
50	12000	13333	0.88	0.98	0.10	9.77	13.5	3.39
50	14500		1.06				-5.19
50	13500		0.99				1.87
Concentration [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	49000	50333	1.15	1.29	0.13	10.2
Control 2	57500		1.41
Control 3	44500		1.31
1	47500	45167	1.19	1.19	0.06	5.12	8.12	7.57
1	47500		1.26				2.59
1	40500		1.14				12.0
2.5	39000	43833	1.02	1.30	0.27	20.6	20.7	-0.78
2.5	47500		1.56				-20.7
2.5	45000		1.32				-2.35
5	64500	63833	1.68	1.65	0.43	25.9	-30.2	-27.5
5	82000		2.06				-59.2
5	45000		1.20				6.77
10	48000	50000	1.07	1.42	0.36	25.6	17.3	-9.59
10	48000		1.39				-7.35
10	54000		1.79				-38.7
25	38500	43167	1.05	1.13	0.38	33.8	18.9	12.2
25	52000		1.55				-20.3
25	39000		0.80				38.0
50	17000	14667	0.35	0.08	0.30	402	73.0	87.7
50	16500		0.13				90.0
50	10500		0.00 *				100
Concentration [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	150000	149833	1.12	1.09	0.22	19.9
Control 2	136500		0.86
Control 3	163000		1.30
1	127500	136333	0.99	1.11	0.12	11.0	9.73	-1.14
1	143000		1.10				-0.75
1	138500		1.23				-12.4
2.5	119000	142667	1.12	1.18	0.05	4.62	-1.98	-7.52
2.5	156500		1.19				-9.00
2.5	152500		1.22				-11.6
5	101500	104000	0.45	0.52	0.24	46.9	58.6	52.8
5	112000		0.31				71.5
5	98500		0.78				28.4
10	75500	76667	0.45	0.42	0.20	47.5	58.6	61.4
10	88000		0.61				44.6
10	66500		0.21				81.0
25	42000	53000	0.09	0.20	0.23	116	92.1	81.6
25	54500		0.05				95.7
25	62500		0.47				56.9
50	21000	18500	0.21	0.25	0.27	109	80.7	77.1
50	16500		0.00				100
50	18000		0.54				50.7

* The number of cells/mL was lower at observation time 48 h compared to 24 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)

Concen-tration [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	150000	1.13	1.13	0.03	2.61
Control 2	136500	1.10
Control 3	163000	1.16
1	127500	1.08	1.10	0.02	1.79	4.67	2.74
1	143000	1.12				1.30
1	138500	1.11				2.24
2.5	119000	1.06	1.11	0.05	4.52	6.70	1.58
2.5	156500	1.15				-1.36
2.5	152500	1.14				-0.60
5	101500	1.00	1.01	0.02	2.22	11.4	10.7
5	112000	1.04				8.49
5	98500	0.99				12.3
10	75500	0.90	0.91	0.05	5.15	20.1	19.8
10	88000	0.96				15.6
10	66500	0.86				23.8
25	42000	0.71	0.78	0.07	8.60	37.4	30.9
25	54500	0.80				29.7
25	62500	0.84				25.7
50	21000	0.48	0.43	0.04	9.37	57.8	61.6
50	16500	0.40				64.9
50	18000	0.43				62.3

There was a slight apparent inhibition of the average specific growth rate [µ] at the test concentrations of 1 mg/L and 2.5 mg/L over the whole testing period 0-72 h, which is considered to be casual. A definite inhibition of the average specific growth rate [µ] could be observed at the test concentrations of 5, 10, 25 and 50 mg/L over the whole testing period 0-72 h.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1) Biomass in controls increased 30x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 16.1 % (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.6% (nom. value ≤ 7%).