Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons

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:

21 July – 03 Sep 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:

Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons

EC Number:

701-305-8

Molecular formula:

not applicaple - UVCB

IUPAC Name:

Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons

Test material form:

solid: pellets

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).

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 pitch material was reduced to small pieces with a pestle and then was directly weighed into the glass 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, 50, 100, 250, 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, 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:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

no data specified: Compositions of stock solutions given in the report

Test temperature:

25 +/- 2°C

pH:

Without algae: initial: 8.0 (control), 7.9 (loading rate 100 mg/L); 72 h: 7.9 (control), 8.0 (loading rate 100 mg/L)
With algae: initial: 8.0 (control), 8.0 (loading rate 100 mg/L); 72 h: 8.6 (control), 8.7 (loading rate 100 mg/L)

Salinity:

no data specified: Compositions of stock solutions given in the report

Nominal and measured concentrations:

Time [h] Control 100 mg/L loading
====================================
0 0.6 3.0 mg C/ml (TOC of WAFs)
72 2.8 2.9 mg C/mL (TOC of WAFs)
====================================

TOC-values confirmed the low water solubility of the test item.
An increase of the TOC-value could be observed in the control during 72 h. The TOC-value remained approximately stable (96.6 %) at
test loading rate of 100 mg/L. The increase of the TOC-value in the control is probably caused by organic components derived from
the metabolismof the algae.

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

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 illumination
- 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

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Growth curves of control and treated algae were comparable.

Reported statistics and error estimates:

not applicable

Any other information on results incl. tables

The tables are excerpts from the original report

Table 3: Loading rate 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

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	18000	20000	1.28	1.34	0.35	25.9
Control 2	29500		1.77
Control 3	16000		1.16
Control 4	11000		0.79
Control 5	21000		1.44
Control 6	24500		1.59
100	15000	17900*	1.10	1.26	0.17	13.5	17.9	5.62
100	23000		1.53				-14.0
100	17500		1.25				6.4
100	18500		1.31				2.3
100	26000*		-				-
100	15500		1.13				15.5
	48 h
Control 1	78500	100083	1.47	1.61	0.16	9.99
Control 2	116500		1.37
Control 3	95000		1.78
Control 4	55500		1.62
Control 5	114500		1.70
Control 6	140500		1.75
100	108500	90000*	1.98	1.62	0.22	13.8	-22.5	-0.28
100	93500		1.40				13.2
100	76000		1.47				9.06
100	91000		1.59				1.34
100	124500*		-				-
100	81000		1.65				-2.41
	72 h
Control 1	211500	245583	0.99	0.94	0.24	25.74
Control 2	248500		0.76
Control 3	244500		0.95
Control 4	220000		1.38
Control 5	261000		0.82
Control 6	288000		0.72
100	297500	237600*	1.01	0.96	0.14	14.0	-7.82	-3.00
100	238000		0.93				0.13
100	180000		0.86				7.83
100	210500		0.84				10.4
100	547500*		-				-
100	262000		1.17				-25.5

Remark: * Results of cells/mL of replicate 5 of test loading rate 100 mg/L were identified as outliers according to Grubbs Test and were excluded from calculation of the mean value of cells/mL, calculation of the growth curves, average specific growth rate and inhibition of the average specificic growth rate.

Table 4: 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

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	211500	1.25	1.30	0.04	2.90
Control 2	248500	1.30
Control 3	244500	1.30
Control 4	220000	1.26
Control 5	261000	1.32
Control 6	288000	1.35
100	297500	1.36	1.28	0.08	6.55	-5.07	1.10
100	238000	1.29				0.67
100	180000	1.19				7.85
100	210500	1.25				3.83
100	547500*	-				-
100	262000	1.32				-1.80

Remark: * Results of cells/mL of replicate 5 of test loading rate 100 mg/L were identified as outliers according to Grubbs Test and were excluded from calculations of the average specificic growth rate and inhibition of the average specificic growth rate.

Table 5: Loading rate of the test item, cells/mL in the test flasks at 72 h, yield over the whole test period 0 -72 h, average of yield, standard deviation and coefficient of variation V_c [%], inhibition [%] and average of inhibition of yield [%]

Loading rate [mg/L]	Cells/mL	Yield Cells/mL	Yield Cells/mL Mean value	Standard deviation	Coefficient of variation Vc [%]	Inhibition of Yield [%]	Inhibition of Yield [%] Average
	72 h
Control 1	211500	206500	240583	27795	11.6
Control 2	248500	243500
Control 3	244500	239500
Control 4	220000	215000
Control 5	261000	256000
Control 6	288000	283000
100	297500	292500	232600	45375	19.5	-21.6	3.32
100	238000	233000				3.15
100	180000	175000				27.3
100	210500	205500				14.6
100	547500*	542500*				-
100	262000	257000				- 6.82

Remark: * Results of cells/mL of replicate 5 of test loading rate 100 mg/L were identified as outliers according to Grubbs Test and were excluded from calculations of yield and inhibition of yield.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item caused no inhibition of the average specific growth rate over the whole testing period 0-72 h and during observation
intervals 0-24 h, 24-48 h and 48-72 h.
The test item caused no inhibition of yield during test period 0-72 h.