Heptanal, oligomeric reaction products with aniline

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:

28 October 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables" for details.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables" for details.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables" for details.

Principles of method if other than guideline:

See "Any other information on materials and methods incl. tables" for details.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The samples were analysed on the day of sampling and were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.

Vehicle:

not specified

Details on test solutions:

Preparation of test solutions
The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring after which the resulting mixture was allowed to stabilize for 2 to 4 hours (combined limit/range-finding test: 3½h, range-finding test: 2¾h, final test: 3h). Subsequently, the Saturated Solution (SS) was siphoned off through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Not specified

Post exposure observation period:

Not specified

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Hepteen Base®;
%SS prep. at 100 mg/L Measured concentration (mg/L) TWA (mg/L)
t=0h t=24h t=72h
0.032 0.00209 0.000080 0.00039 0.00025
0.1 0.00677 0.00016 0.00189 0.00071
0.32 0.0233 0.0140 0.00601 0.012
0.321 0.0224 0.0020 0.00057 0.0029
1.0 0.0760 0.0384 0.0251 0.039
3.2 0.218 0.264 0.130 0.20
10 0.660 0.820 0.433 0.64

Details on test conditions:

Fresh water algae culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessel: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL
Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 89 µE/m2/s.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous
shaking.

Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test, microscopic observations were
performed on the control and 0.32% SS to observe for any abnormal appearance of the algae.

Recording of cell densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

Test concentrations
Hepteen Base®: Solutions containing 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of a SS prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of the control and each test concentration for sampling purposes after 24 hours, 1 replicate of each test concentration without algae.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 ng/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between 20 to 21 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between 13 to 18 µg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between 10 - 11

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

13 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between 13-14

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between 2.9 - 5.5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between 4.9 - 8.1

Details on results:

The results of a preceding combined limit/range-finding test were considered inconclusive and rejected. The growth rates in the control group were very low and varied strongly and as such, the results of the test are not reported and the test was repeated as a range-finding test.

Range-finding test
The mean cell densities measured during the range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.
Inhibition of growth rate and yield was insignificant at the lowest test group (0.10% SS), significant at the test group containing 1.0% SS and near to completely inhibited at 10 and 100% of the SS.

Final test
Measured test item concentrations
At the start of the test, the actual test concentrations were 0.0021, 0.0068, 0.023, 0.076, 0.22 and 0.66 in 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at 100 mg/L. These concentrations did not remain stable during the test duration, i.e. were at 2.5-66% of initial at the end of the test. Thus, time weighted average (TWA) concentrations were calculated (see Table 4) and used to determine the effect concentrations. The estimated TWA concentrations were 0.25, 0.71, 12, 39, 200 and 640 µg/L in 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of the SS, respectively.

The determined concentrations in the two highest test groups might, however, be underestimated at the start and the end of the test period. The respective quality control samples were below the specified limits (i.e. were at ~55 to 66% and thus below 70%). Based on the biological responses in the test, it was consequently decided to omit the two highest test groups during the subsequent data analysis. The biological results from those test groups are not relevant for the determination of effect concentrations.
Note, that a small response was detected in the control. However, it was considered to derive from carry-over in the analytical system.

Inhibition of growth rate and inhibition of yield
Growth rates were in the range of the controls at the concentrations from 0.25 to 12 µg/L during the 72-hour test period, whereas the growth rate of algae exposed to 39 µg/L and higher were fully inhibited. Statistically significant inhibition of growth rate was found at average test concentrations of 39 µg/L and higher.

Inhibition of yield increased with increasing concentration of Hepteen Base® resulting in 100% inhibition at 39 µg/L and above. Statistically significant inhibition of yield was found at average test concentrations of 39 µg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 12 µg/L when compared to the control

Experimental conditions
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units).

During the exposure period, the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95% confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Mean cell densities (x10⁴cells/mL) during the range-finding test

Time (h)	Hepteen Base®; % SS prep. at 100 mg/L
	Control	0.10	1.0	10	100
0	1.0	1.0	1.0	1.0	1.0
72	41.8	38.0	7.6	1.1	1.0

 

Percentage inhibition of growth rate during the range-finding test

Hepteen Base® % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.230	0.1174	3
0.10	1.199	0.1140	3	2.5
1.0	0.642	0.1842	3	47.8
10	0.030	0.0526	3	97.5
100	0.000	0.0000	3	100.0

 

Percentage inhibition of yield during the range-finding test

Hepteen Base® % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	40.8	15.47	3
0.10	37.0	13.45	3	9.3
1.0	6.6	4.01	3	83.9
10	0.1	0.18	3	99.7
100	0.0	0.00	3	100.0

 

Percentage inhibition of growth rate (total test period) during the final test

Hepteen Base® TWA conc. (µg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.663	0.0145	6
0.25	1.652	0.0125	3	0.7
0.71	1.639	0.0169	3	1.4
12	1.569	0.0701	3	5.7
39	0.000	0.0000	3	100.0*

* - effect was statistically significant

 

Percentage inhibition of growth rate at different time intervals during the final test

Hepteen Base® TWA conc. (µg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.675		1.779		1.536
0.25	3	1.714	-2.3	1.736	2.4	1.507	1.9
0.71	3	1.593	4.9	1.811	-1.8	1.514	1.4
12	3	1.538	8.2	1.697	4.6	1.473	4.1
39	3	0.000	100.0	0.472	73.5	-0.472	130.7

Percentage inhibition of yield during the final test

Hepteen Base® TWA conc. (µg/L)	Mean	Std. Dev.	n	%Inhibition
Control	146.1	6.38	6
0.25	141.3	5.29	3	3.3
0.71	135.9	6.82	3	7.0
12	111.4	24.17	3	23.7
39	0.0	0.00	3	100.0*

* - effect was statistically significant

Effect parameters

	Parameter (µg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	12	10	13	20
	lower 95%-cl		10	13	20
	upper 95%-cl		11	14	21
Yield	Value	12	4.2	6.5	15
	lower95%-cl		2.9	4.9	13
	upper 95%-cl		5.5	8.1	18

 

pH levels recorded during the final test

Hepteen Base® TWA conc. (µg/L)	pH
	t=0h	t=72h
Control	8.1	8.4
0.25	8.0	8.5
0.71	8.0	8.4
12	8.0	8.3
39	8.0	8.1
200	8.0	8.1
640	7.9	8.1

 

Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.475	0.0948	3
0.18	1.421	0.1100	3	3.7
0.32	1.182	0.3745	3	19.9
0.56	1.221	0.0545	3	17.2
1.0	1.080	0.0239	3	26.8
1.8	0.432	0.0521	3	70.7
3.2	0.227	0.0183	3	84.6

Overview of % inhibition of yield in the reference test:

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	84.8	23.34	3
0.18	72.5	21.61	3	14.5
0.32	46.8	35.15	3	44.8
0.56	38.3	6.67	3	54.8
1.0	24.6	1.81	3	71.0
1.8	2.7	0.56	3	96.8
3.2	1.0	0.11	3	98.8

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Hepteen Base® reduced the growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 39 µg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 20 µg/L with a 95% confidence interval ranging from 20 to 21 µg/L.
The EC50 for yield inhibition (72h-EYC50) was 15 µg/L with a 95% confidence interval ranging from 13 to 18 µg/L.
The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 µg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Hepteen Base®.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266 and the OECD series on testing and assessment number 23, 2000.

The batch of Hepteen Base® tested was aclear amber liquid with a purity of 99.7% and not completely soluble in test mediumat the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring after which the resulting mixture was allowed to stabilize for approximately 3 hours. Subsequently, the Saturated Solution (SS) was siphoned off and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

A final test was performed based on the results of a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of a SS prepared at a loading rate of 100 mg Hepteen Base® per litre.

The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the actual test concentrations were 0.0021, 0.0068, 0.023, 0.076, 0.22 and 0.66 mg/L in 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at 100 mg/L. These concentrations did not remain stable during the test duration, i.e. were at 2.5-66% of initial at the end of the test. Thus, the time weighted average (TWA) concentrations were calculated and used to determine the effect concentrations. The estimated TWA concentrations were 0.25, 0.71, 12, 39, 200 and 640 µg/L in 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of the SS, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

Under the conditions of the present study with Pseudokirchneriella subcapitata, Hepteen Base® reduced the growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 39 µg/L and higher.

The EC₅₀for growth rate inhibition (72h-E_RC₅₀) was 20 µg/L with a 95% confidence interval ranging from 20 to 21 µg/L.

The EC₅₀for yield inhibition (72h-E_YC₅₀) was 15 µg/L with a 95% confidence interval ranging from 13 to 18 µg/L.

The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 µg/L.

Description of key information

Under the conditions of the present study with Pseudokirchneriella subcapitata, Hepteen Base®  reduced the growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 39 µg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 20 µg/L with a 95% confidence interval ranging from 20 to 21 µg/L.

The EC50 for yield inhibition (72h-EYC50) was 15 µg/L with a 95% confidence interval ranging from 13 to 18 µg/L.

The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 µg/L.

In accordance with Regulation (EC) 1272/2008 Hepteen Base® meets the criteria for Acute Aquatic Toxicity Category 1 and Chronic Aquatic Category 1.

Key value for chemical safety assessment

EC50 for freshwater algae:

15 µg/L

EC10 or NOEC for freshwater algae:

12 µg/L

Additional information

Study was conducted in accordance with the Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

In addition, the procedures were designed to meet the test methods prescribed by the following guideline and guidance document:

•       Council regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C3: “Algal Inhibition Test”; Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.

•       Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000

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