Petroleum resins

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16. Apr. 2018 to 22. Jun. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The real cell concentration at the beginning of each test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
From these mixtures samples for the analytical determination were taken, afterwards the pH-value was measured.

Vehicle:

no

Details on test solutions:

A water-accommodated fraction (WAF) was prepared for the test. This was done by weighing each nomi-nal load (1 / 3.2 / 10 / 32 / 100 mg/L), adding the corresponding amounts of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 23 hours (experiment I), respectively 24 hours (experiment II). The resulting solutions were filtrated through 0.45 µm PTFE filters.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification: Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae
Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observtaion period specified in the study report.

Hardness:

Not specified

Test temperature:

21.8 – 23.5°C (experiment I)
21.6 – 22.8°C (experiment II)

pH:

7.6 - 8.4

Dissolved oxygen:

Not specified

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration

Details on test conditions:

Pre-Culture
Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions(5000 lux, 20.6 - 23.5°C in experiment I and 21.4 – 22.4°C in experiment II). The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of cul-ture was determined.

Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.363 mL experiment I, 0.349 mL experiment II) to achieve a cell concentration of 2.3 *103 cells/mL (experiment I) and 2.0 *103 cells/mL (experiment II). For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.635 mL experiment I, 0.610 mL experiment II).
The real cell concentration at the beginning of each test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
From these mixtures samples for the analytical determination were taken, afterwards the pH-value was measured.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 ± 1 hours, the cell number was determined with an electronic particle counter. After the test, samples for the analytical determination were taken, afterwards the pH value in treatments and blank control was measured again.
At the end of each test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of each experiment.

Number of replicates: 6 replicates for the blank control; 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: 5000 lux
Temperature: 21.8 – 23.5°C (experiment I); 21.6 – 22.8°C (experiment II)
Replicates (Blank control): 45 ±1 mL algal medium and algae
Replicates (Treatments): 45 ±1 mL test solution and algae

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate & Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate & Yield

Details on results:

The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each repli-cate was determined by measuring the cell numbers every 24 ± 1 hours with an electronic particle coun-ter. Growth rate µ and the yield were determined from the cell number at the respective observation times.
In both experiments scattering inhibition values were visible and therefore no dose-response relationship. The results can therefore only be specified as a range. Statistical evaluation was performed only with the lowest treatment to demonstrate significant inhibition.
At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.
The measured DOC values are all in the same very low range as the blank value. Nevertheless, the inhibition of algal growth in all treatments showed the presence of dissolved test item in the test solutions.
Therefore, the results refer to the nominal values.
The pH of the blank control should not fluctuate by more than 1.5 units. The change in the blank control was 0.1 units in experiment I and 0.6 units in experiment II, respectively.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Results with reference substance (positive control):

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estima-tion of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.
In both experiments the scattered inhibition values were apparent and therefore no dose-response rela-tionship could be established. The results can therefore only be specified as a range. Statistical evaluation was performed only with the lowest treatment (experiment II) to demonstrate significant inhibition for determination of the NOEC

Biological Results of K₂Cr₂O₇

Parameter	Value	95% confidence interval
72h EC50	0.90 mg/L	0.77 – 1.1 mg/L
72h EC50	0.42 mg/L	0.35 - 0.51 mg/L

 

Findings

Cell Numbers

The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

 

Cell Number/mL (experiment I)

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2340	9427	61030	276953
Blank control	SD	0	1067	4114	15317
1	Mean	2340	7047	50187	228773
1	SD	0	501	7669	26534
3.2	Mean	2340	7300	35140	174647
3.2	SD	0	1087	7831	24831
10	Mean	2340	9267	52027	221613
10	SD	0	1090	3098	35878
32	Mean	2340	8833	61193	276173
32	SD	0	480	3391	25685
100	Mean	2340	7513	41667	201140
100	SD	0	477	6637	25942

Cell Number/mL (experiment II)

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2000	8877	68367	355470
Blank control	SD	0	1184	9391	46991
1	Mean	2000	8813	48700	217700
1	SD	0	1000	10122	33247
3.2	Mean	2000	8427	58920	196393
3.2	SD	0	691	1542	2278
10	Mean	2000	7773	64507	236553
10	SD	0	408	12965	19086
32	Mean	2000	7653	45293	190827
32	SD	0	945	8512	20139
100	Mean	2000	8300	61893	230407
100	SD	0	583	3551	12373

SD = Standard Deviation

 

Light Intensity

In the following table, the light intensity during the tests is stated:

 

Light Intensity (both experiments)

	0 h	24 h	48 h	72 h
Light intensity [Lux]	5000	5000	5000	5000

 

pH values

In the following table, the pH values measured at the start and the end of the tests are stated:

 

pH values (experiment I)

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.8	7.7
1	7.7	7.7
3.2	7.7	7.5
10	7.6	7.6
32	7.7	7.9
100	7.7	7.8

pH values (experiment II)

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.8	8.4
1	7.9	7.9
3.2	8.1	7.9
10	8.1	7.9
32	8.1	7.8
100	8.1	7.8

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the tests is stated:

 

Microscopical Observations (both experiments)

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
1	Yes
3.2	Yes
10	Yes
32	Yes
100	Yes

 

Analytical Determination

At the start and at the end of each test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

Since the measured DOC values are all in the same very low range as the blank value, the results refer to the nominal values.

The details are given in the following tables:

 

Measured Concentrations IC and TC (experiment I)

Nominal Concentration Test Item	Measured TC  t = 0 h	Measured TC  t = 72 h	Measured IC  t = 0 h	Measured IC  t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	6.71	10.42	6.74	5.49
1	6.83	8.71	6.88	5.48
3.2	6.87	8.72	6.91	6.13
10	6.41	11.18	6.38	5.69
32	6.79	10.15	6.85	4.92
100	6.78	8.87	6.78	5.86

LOQ (Limit of quantification) TC = 4.07 mg/L

LOQ (Limit of quantification) IC = 1.34 mg/L

 

Measured Concentrations IC and TC (experiment II)

Nominal Concentration Test Item	Measured TC  t = 0 h	Measured TC  t = 72 h	Measured IC  t = 0 h	Measured IC  t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	7.18	5.82	6.94	5.13
1	7.02	5.86	6.72	5.32
3.2	6.63	6.25	6.48	5.89
10	7.31	6.16	7.14	5.79
32	7.09	6.52	6.85	6.29
100	6.93	6.21	6.83	5.67

LOQ (Limit of quantification) TC = 2.08 mg/L

LOQ (Limit of quantification) IC = 1.27 mg/L

 

Measured Concentrations DOC (experiment I)

Nominal Concentration Test Item	Measured DOC (TC-IC)  t = 0 h	Measured DOC (TC-IC)  t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.00	4.93	--	--
1	0.00	3.23	0.00	-1.70
3.2	0.00	2.59	0.00	-2.34
10	0.03	5.48	0.03	0.55
32	0.00	5.22	0.00	0.29
100	0.00	3.01	0.00	-1.92

Measured Concentrations DOC (experiment II)

Nominal Concentration Test Item	Measured DOC (TC-IC)  t = 0 h	Measured DOC (TC-IC)  t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.23	0.68	--	--
1	0.30	0.54	0.06	-0.15
3.2	0.16	0.36	-0.08	-0.33
10	0.18	0.37	-0.06	-0.32
32	0.24	0.23	0.01	-0.46
100	0.09	0.54	-0.14	-0.15

 

Calculated Test item Concentrations (experiment I)

Nominal Concentration Test Item	Calculated Concentration Test Item  t = 0 h	Calculated Concentration Test Item  t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	%	%
Blank control	--	--	--	--
1	0.0	-2.0	0	-196
3.2	0.0	-2.7	0	-84
10	0.0	0.6	0	6
32	0.0	0.3	0	1
100	0.0	-2.2	0	-2

Calculated Test item Concentrations (experiment II)

Nominal Concentration Test Item	Calculated Concentration Test Item  t = 0 h	Calculated Concentration Test Item  t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	%	%
Blank control	--	--	--	--
1	0.1	-0.2	7	-17
3.2	-0.1	-0.4	-3	-12
10	-0.1	-0.4	-1	-4
32	0.0	-0.5	0	-2
100	-0.2	-0.2	0	0

 

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

 

Growth Rate µ, Yield (experiment I)

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.59	274613
	SD	0.02	15317
1	Mean	1.53	226433
	SD	0.04	26534
3.2	Mean	1.44	172307
	SD	0.05	24831
10	Mean	1.51	219273
	SD	0.05	35878
32	Mean	1.59	273833
	SD	0.03	25685
100	Mean	1.48	198800
	SD	0.04	25942

SD = Standard Deviation

 

Growth Rate µ, Yield (experiment II)

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.72	353470
	SD	0.05	46991
1	Mean	1.56	215700
	SD	0.05	33247
3.2	Mean	1.53	194393
	SD	0.00	2278
10	Mean	1.59	234553
	SD	0.03	19086
32	Mean	1.52	188827
	SD	0.04	20139
100	Mean	1.58	228407
	SD	0.02	12373

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

 

Inhibition Values (experiment I)

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1	4.07	17.54
3.2	9.78	37.25
10	4.82	20.15
32	0.09	0.28
100	6.79	27.61

Inhibition Values (experiment II)

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1	9.48	38.98
3.2	11.31	45.00
10	7.76	33.64
32	11.94	46.58
100	8.24	35.38

 

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Petroleum Resins (Kendex 0897) were determined:

Results of the test item
Endpoint NOEC LOEC EC10 EC50
Growth Rate < 1 mg/L n.d. n.d. > 100 mg/L
Yield < 1 mg/L n.d. n.d. > 100 mg/L
n.d.: not determinable due to lack of dose-response relationship

Executive summary:

Determination of the toxicity of Petroleum Resins (Kendex 0897) against Desmodesmus subspicatus according to OECD 201 resp. EU C.3

 

Findings and Results:

Two valid experiments were performed. Due to the large scattering of the results, a second experiment was carried out in the same way as the first experiment. The results of both experiments are stated in this report. However, the biological results are only related to the second experiment.

The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ± 1 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

In both experiments scattered inhibition values were visible and therefore no dose-response relationship was observed. The results can therefore only be specified as a range. Statistical evaluation was performed only with the lowest treatment to demonstrate significant inhibition.

 

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

The measured DOC values are all in the same very low range as the blank value. Nevertheless, the inhibition of algal growth in all treatments showed the presence of dissolved test item in the test solutions.

Therefore, the results refer to the nominal values.

 

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test item Petroleum Resins (Kendex 0897) were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	< 1 mg/L	n.d.	n.d.	> 100 mg/L
Yield	< 1 mg/L	n.d.	n.d.	> 100 mg/L

n.d.: not determinable due to lack of dose-response relationship

Description of key information

The following results for the test item Petroleum Resins (Kendex 0897) were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	< 1 mg/L	n.d.	n.d.	> 100 mg/L
Yield	< 1 mg/L	n.d.	n.d.	> 100 mg/L

n.d.: not determinable due to lack of dose-response relationship

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

Two valid experiments were performed. Due to the large scattering of the results, a second experiment was carried out in the same way as the first experiment. The results of both experiments are stated in this report. However, the biological results are only related to the second experiment.

The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ± 1 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

In both experiments scattered inhibition values were visible and therefore no dose-response relationship was observed. The results can therefore only be specified as a range. Statistical evaluation was performed only with the lowest treatment to demonstrate significant inhibition.

 

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

The measured DOC values are all in the same very low range as the blank value. Nevertheless, the inhibition of algal growth in all treatments showed the presence of dissolved test item in the test solutions.

Therefore, the results refer to the nominal values.

 

The following results for the test item Petroleum Resins (Kendex 0897) were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	< 1 mg/L	n.d.	n.d.	> 100 mg/L
Yield	< 1 mg/L	n.d.	n.d.	> 100 mg/L

n.d.: not determinable due to lack of dose-response relationship