Fast Pyrolysis Bio-oil

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2013 – 04 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all concentrations and from the control at test start (t = 0 hours) and after 24, 48 and 72 hours. All test item concentrations were analysed at 0 and 72 hours to verify test concentrations.

Vehicle:

no

Details on test solutions:

For the preparation of the test item solutions, the test item was stirred until a homogeneous consistency was obtained. Subsequently an appropriate amount of the test item was directly weighed onto teflon tape and given to test medium and stirred slightly for 48 hours, protected from light, for each individual loading rate. Afterwards the undissolved test item was allowed to sediment and/or float for a period of 24 hours. The test item solution used in the test vessel was withdrawn by suction from the middle of the preparation vessel (according to OECD SERIES ON TESTING AND ASSESSMENT No. 23). Transfer of undissolved test item was avoided. Each solution was applied to the respective test vessel.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Strain: SAG 86.81. The alga used in this study were purchased from the Culture Collection of Algae (SAG), Albrecht-von-Haller-Institut, Nikolausberger Weg 18, 37073 Göttingen, Germany. The algae were grown semi-continuously under sterile conditions in the laboratory. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

not determined

Test temperature:

21.3 –22.5 °C

pH:

7.29 – 8.68

Dissolved oxygen:

not determined

Salinity:

not determined

Nominal and measured concentrations:

nominal test concentrations: 100, 31.3, 9.77, 3.05, 0.954 mg/L (loading rate of the WAF); all five test concentrations were analysed resulting in measured concentrations of 38.0mg/ (100 mg/L nominal), 11.6 mg/L (31.3 mg/L nominal), 2.25 mg/L (9.77 mg/L nominal) and 0.783 (3.05 mg/L nominal) which corresponds to 38% rsp. 37%, 23% and 26% of the nominal loading rate. The lowest concentrations were found to be below Limit of Quantification (2 mg/L). The mean initial measured content of Fast Pyrolysis Bio-oil was 42 % of nominal. After 72 h the mean measured concentration of Fast Pyrolysis Bio-oil in the test solutions was 23% of nominal concentration.

Details on test conditions:

500 mL Erlenmeyer flasks with aluminium caps and two baffles; Continously shaking on a rotating shaker; Continuously illuminated from the top, approx. 4800 – 5300 lux; Initial cell density 0.5 x 10e4 cells/mL; Volume of test solution 150 - 200 mL

Reference substance (positive control):

yes

Remarks:

potassium dichromate (ErC50 0.859 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

93.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 83.2 - 105 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 55.1 – 63.8 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 31.63 – 39.71 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 20.92 – 24.23 mg/L

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Details on results:

Cell number and growth rates increase with the lower loading rates up to 9.77 mg/L (nominal). With the second highest loading rate of 31.3 mg/l almost no effect was obeserved. With the highest loading rate of 100 mg/L (nominal) a significant inhibition of algal growth was found.

Results with reference substance (positive control):

EC50 growth rate 0.859 mg/L
EC50 yield 0.238 mg/L
EC50 biomass 0.365 mg/L
NOEC 0.0741 mg/L
LOEC 0.222 mg/L

Reported statistics and error estimates:

The statistical evaluation for 3 days was performed for growth rate and yield using SAS® (2002–2008). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was performed by using the Levene-test. The NOEC and LOEC were determined by using the Dunnett´s t-test for growth rate and Welch Bonferroni-Holms corrected for yield.
The EC50-value for growth rate and yield was calculated using Spearman-Kärber estimator (ANELLIS & WERKOWSKY, 1968). Due to statistical reasons the inhibition values of 31.3 and 100 mg/L were rounded and included in the calculation. EC10, 20 was not calculable.

Table:       Average cell number for each sampling time and concentration

Test item conc. loading rate [mg/L]	Average cell numbers/mL^1)
	0d	1d	2d	3d
Control	0.5	2.25	8.97	33.40
0.954	0.5	2.36	10.68	40.17
3.05	0.5	2.48	12.16	56.25
9.77	0.5	2.84	13.37	60.94
31.3	0.5	2.11	7.45	32.28
100	0.5	2.00	2.62	3.23

¹⁾Algae counts are divided by 10000. At the start, the cell density was adjusted to 0.5 × 10⁴cells/mL

Table:   Percentage inhibition of growth rate

Test item conc. loading rate[mg/L]	Inhibition of growth rate [%]
	0-1d	0-2d	0-3d
Control	0.0	0.0	0.0
0.954	-3.3	6.2	-4.5
3.05	-6.7	-10.8	-12.4
9.77	-15.8	-14.0	-13.6
31.3	4.4	6.3	0.4
100	7.6	42.5	55.6*

Table:       Percentage inhibition of yield

Test item conc. loading rate[mg/L	Inhibition of yield [%]
	0-1d	0-2d	0-3d
Control	0.0	0.0	0.0
0.954	-6.3	-20.2	-20.6
3.05	-13.1	-37.7	-69.5
9.77	-33.7	-51.9	-83.7
31.3	8.0	17.9	3.4
100	14.3	75.0	91.7*

*statistically significant different to the control

Validity criteria fulfilled:

yes

Conclusions:

Short-term toxicity of fast pyrolysis bio-oil (water accommodated fraction) to Desmodesmus subspicatus was tested in a 72 h growth inhibition study. Statistically significant adverse effects were observed at the two highest loading rates (31.3 and 100 mg/L), and the determined NOEC and ErC50 for the fresh water algae were 11.6 and 35.4 mg/L, respectively (analyzed concentrations). Therefore, the test material showed slight acute toxicity to D. subspicatus.

Executive summary:

Short-term toxicity of fastpyrolysis bio-oil (water accommodated fraction) to an aquatic algae Desmodesmus subspicatus was determined in a GLP compliant growth inhibition study according to OECD Guideline no 201 (2011) "Alga, Growth Inhibition Test". All validity criteria of the guideline were fulfilled.

 

Initial concentration of 0.5 x 10⁴  algal cells/mL in each test vessel were exposed in a static test system for 72 hours. After performing a range-finding test, the main test was performed with test item concentrations of 0.954, 3.05, 9.77, 31.3 and 100 mg/L loading rate. Six replicates were run for the control and three for each test item concentrations. In addition, four replicates were run in parallel for analytical determination.

 

After 24, 48 and 72 h, the cell dencity was determined by fluorescence detection and growth curves for each test concentration were produced. At the end of the test perioid,  EC_{10, 20, 50} LOEC and NOEC values for growth and yield were evaluated. All test item concentrations were analyzed at 0 and 72 h to verify the actual test concentrations.

 

The EC₅₀ values for growth rate were determined to be 93.3 mg/L loading rate (nominal) and 35.4 mg/L loading rate (actual). The EC₅₀ values for yield were determined to be 59.2 mg/L loading rate (nominal) and 22.5 mg/L loading rate (actual) The actual concentrations were based on the geometric mean of each test item concentration.

The LOEC values for growth and yield were determined to be 31.3 mg/L loading rate (nominal) and 11.6 mg/L loading rate (actual) and the NOEC values were determined to be 100 mg/L loading rate (nominal) and 38.0 mg/L loading rate (actual). The actual concentrations were based on the geometric mean of each test item concentration.

 

The test material showed slight toxicity to D. subspicatus as the determined 72 h ErC₅₀ value (growth) was between 10 and 100 mg/L.

 

The study is reliable without restriction.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Full documentation not available due to type of information (publication)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Details on sampling:

The number of algal cells in the flasks was determined by counting under the microscope (Nikon Eclipse E600, 400x) with a Burker haemocytometer at the beginning and at the end of the test.

Vehicle:

no

Details on test solutions:

Stock solutions (10 g/L) of the bio-oil was prepared in the OECD growth medium (pH 8.1). There were control and five test concentrations. Concentration range: 18–60 mg/L.
Volume: 70 mL

Test organisms (species):

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

Details on test organisms:

Unicellular green alga, origin: SAG Goettingen University.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

20 +/1 1°C

pH:

8.1

Nominal and measured concentrations:

Control and five test concentrations. Concentration range: 18–60 mg/L

Details on test conditions:

The test was performed in In 100 mL borosilicate glass flasks with 70 mL of test solution. There were a control and five test concentrations. The 70 mL samples were inoculated with an aliquot of exponentially growing algae to ensure an algal concentration of 10^4 cells /mL and then incubated for 72 hours.

Illumination: continuous light, 4000 K ,6000–8000 lx.
Other incubation conditions: Continually shaken at 100 rpm (orbital shaker).

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 38 mg/L

Conc. based on:

test mat.

Reported statistics and error estimates:

n = 3, 95 % confidence limits: lower = 31 and upper = 45

Conclusions:

Fast pyrolysis bio-oil from pine wood (BTG) showed slight toxicity to fresh water algae (EC50, 48 h = ca. 38 mg/L).

Executive summary:

The effects of the test material (BTG, commercial pine wood based fast pyrolysis bio-oil) on the growth of freshwater algae Pseudokirchneriella subcapitata was tested according to OECD guideline no. 201 (2006) and in complience with GLP. Five test concentrations were prepared and test flasks with the green algae were incubated for 72 h. The number of algal cells in the flasks was determined at the beginning and at the end of the test, and the average specific growth rate was calculated. The EC50 value was determined. According to the algae growth inhibition test, fast pyrolysis bio oil from pine wood showed slight toxicity to green algae, as the determined 48 h EC50 value was between 10 and 100 mg/L (EC50_algae = 38 mg/L).

The study is reliable with restrictions as full documentation is not available due to type of information (publication).

Description of key information

Short-term toxicity of the test material (Fast Pyrolysis Bio-oil, WAF) to an aquatic algae Desmodesmus subspicatus was determined in a GLP compliant study according to OECD Guidelines for Testing Chemicals (2011) no 201 "Alga, Growth Inhibition Test". Statistically significant adverse effects were observed at the two highest loading rates (31.3 and 100 mg/L), and NOEC and ErC₅₀ were determined at 11.6 and 35.4 mg/L, respectively (analyzed concentrations).

According to a OECD guideline study no. 201 (2006) published in literature, Fast-Pyrolysis Bio-oil from pine wood showed low / slight toxicity to Pseudokirchneriella subcapitata (ErC₅₀ = 38 mg/l).

Key value for chemical safety assessment

EC50 for freshwater algae:

35.4 mg/L

EC10 or NOEC for freshwater algae:

11.6 mg/L

Additional information

Due to poor water solubility of the substance, the organisms were exposed to a Water Accommodated Fraction (WAF) of the test substance. This is a commonly accepted procedure for regulatory studies on poorly water soluble substances.