3,5,8,10,13,15,18,20,23,25,28,30,33,35,38,40,41,43,45,47,51,53,55,57,59,61,63, 65,67,69,71,73-dotriacontazapentacosacyclo [35.3.3.36,7.311, 12.316,17.321,22.326,27.331,32.22,41.236,43.13,40.15,8.110,13.115,18.120,23.125,28.130,33.135,38.145,51.147,73.153,55.157,59.161,63.165,67.169,71] octacontane-44,46,48,49,50,52,54,56,58,60,62,64,66,68,70,72-hexadecone, hydrochloride hydrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of an additional algal species

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Phaeodactylum tricornutum (P. tricornutum) wild type 646 strain were grown in four different mediums as follows: control group (f/2 medium), CB[6] group (f/2 medium with 3 different concentrations of CB[6], CB[7] group (f/2 medium with 3 different concentrations of CB[7], and CB[8] group (f/2 medium with 2 different concentrations of CB[8]. The cell cultures were grown over 11 days and the concentration of algae cells was determinied by optical density (OD) which was measured at 730 nm by plate reader

GLP compliance:

no

Remarks:

study was performed at the Department of Plant Sciences, University of Cambridge, UK

Test organisms (species):

Phaeodactylum tricornutum

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

11 d

Test temperature:

room temperature

Key result

Duration:

11 d

Dose descriptor:

NOEC

Effect conc.:

200 µmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Conclusions:

There was no obvious growth rate change of P. tricornutum at 100 and 200 µM in f/2 medium. Undissolved CB[8] was found under the microscope.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYI/38593-5/2012

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 5.19 - 7.84

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL 1.15 - 1.65

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 1.40 - 2.06

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Observations in the main experiment

Nominal concentrations [mg/L]	Observations
Untreated control	Normal cells
0.15	Chuff cells
0.48	Increased aggregation, chuff and few deformed cells
1.53	Aggregation, chuff and deformed cells
4.88	Aggregation, chuff and very deformed cells
15.63	Chuff cells, occasionally thin cells
50.00	Chuff cells, occasionally thin cells

Growth Rates (m) and Percentage Inhibition of m during the Test Period

NominalConcentration [mg/L]	Growth rate (m) and % inhibition of m
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0589	0.0	0.0609	0.0	0.0593	0.0
0.15	0.0578	1.9	0.0597	1.9	0.0586	1.2
0.48	0.0538	8.7	0.0559	8.2	0.0569	4.2
1.53	0.0538	8.7	0.0540^+	11.4	0.0488*	17.8
4.88	0.0345*	41.4	0.0320*	47.5	0.0323*	45.5
15.63	0.0289*	50.9	0.0229*	62.4	0.0166*	72.0
50.00	0.0096*	83.6	0.0096*	84.2	0.0064*	89.2

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

⁺: at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

NominalConcentration [mg/L]	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	38.0	0.0	288.0	0.0	1348.0	0.0
0.15	36.0	5.3	272.0	5.6	1276.0	5.3
0.48	32.0	15.8	228.0*	20.8	1100.0*	18.4
1.53	32.0	15.8	212.0*	26.4	752.0*	44.2
4.88	16.0*	57.9	76.0*	73.6	232.0*	82.8
15.63	12.0*	68.4	48.0*	83.3	100.0*	92.6
50.00	4.0*	89.5	16.0*	94.4	32.0*	97.6

*   : statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

NominalConcentration [mg/L]	Yield (Y) and % inhibition of Y
	0–72 h
	Y	%
Control	70.7	0.0
0.15	67.0	5.2
0.48	59.0*	16.5
1.53	32.7*	53.8
4.88	9.3*	86.8
15.63	2.3*	96.7
50.00	0.7*	99.1

*   :  statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of CUCURBIT[8]URIL test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly
known as Selenastrum capricornutum), over an exposure period of 72 hours.
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 1.53 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.48 mg/L (nominal) and the LOEC was determined as 1.53 mg/L (nominal). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 0.48 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.15 mg/L (nominal) and the LOEC was determined as 0.48 mg/L (nominal).

Executive summary:

The purpose of the study was to determine the effect of the test item CUCURBIT[8]URIL on the growth of the unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum). Study performed to – OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28^thJuly, 2011. – EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996. – Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal of the European Union L 220 of 24 August 2009).

In conclusion with respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 1.53 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.48 mg/L (nominal) and the LOEC was determined as 1.53 mg/L (nominal). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 0.48 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.15 mg/L (nominal) and the LOEC was determined as 0.48 mg/L (nominal).

Description of key information

CB[8] did not affect algal growth in nutrient supplemented medium at concentrations well above 100mg/L. The effects seen in an OECD 201 guideline study at much lower concentrations are therefore attributed to nutrient depletion.

Additional information

There was no obvious growth rate change of P. tricornutum at 100 and 200 µM in f/2 medium over a testing period of 11 days. Undissolved CB[8] was found under the microscope.

The effect of CUCURBIT[8]URIL test item was also assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 1.53 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.48 mg/L (nominal) and the LOEC was determined as 1.53 mg/L (nominal). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 0.48 – 50.00 mg/L (nominal), therefore the NOEC was determined as 0.15 mg/L (nominal) and the LOEC was determined as 0.48 mg/L (nominal).

The effects are considered to be attributable to nutrient depletion.