1,6-Hexandiammonium dihydroxide, N,N'-bis-(2-hydroxypropyl)-N,N,N',N'-tetramethyl

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:

from 21 OCT 2002 to 24 OCT 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

one validity criterion of updated guideline could not be fulfilled

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

pH-value in water 13-14

Analytical monitoring:

yes

Details on sampling:

- All treatment groups and control
- Sampling: test media at the beginning and end of the test
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Stock solution: 4762 mg/L (2000 mg a.i./L), freshly prepared with test medium
- Dispersion treatment: Agitation
- Equipement: Shaker, SM 25 A (BüHLER)

pH-CONTROL
Three replicates of the highest concentration level were tested as pH control. The stock solution (pH-value = 12.09) was neutralized with 1 N HCL to pH 7.88.

CONTROL
Six replicates (without test item) were tested under the same test conditions as the test replicates

The test item was clearly dissolved in all test replicates throughout the test period.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Name: Desmodesmus subspicatus CHODAT SAG 86.81 (formerly known as Scenedesmus subspicatus)
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Date of receipt: 1997-04-07
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE/m^2s for 24h per day.
- Age of inoculum (at test initiation): a three day old preculture was used as inoculum

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test: Nutrient medium Z according to LÜTTGE et al. (1994).
- Any deformed or abnormal cells observed: not reported


LÜTTGE, U, SCHNEPF, E., LÄUCHLI, A., NAGATA, T. (editors, 1994): Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h algae from the nominal dosage levels 250 - 1000 mg a.i./L and control were transferred to fresh untreated dilution water and allowed to grow for further 4 - 12 d under test conditions.

Test temperature:

22.5 °C - 24.0 °C (min - max); 23.3 °C (mean)

pH:

Treatment groups: 8.14 - 10.82 (min max)
pH control: 8.27 - 8.41 (min max)

Nominal and measured concentrations:

Nominal concentrations: 31.3 - 62.5 - 125 - 250 - 500- 1000 mg a.i./L (75- 149 - 298 - 595 - 1190 - 2381 mg/L test item)
Measured concentrations: see any other information on results

Details on test conditions:

TEST SYSTEM
- Test vessel: Plastic cuvettes (50 mm diameter), transparent, with top, 20 mL
- Fill volume: 10 mL
- Aeration: not reported
- Initial cells density: 8214 cells/mL
- Control end cells density: 400203 [cells/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per pH control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, according to the guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-values at the beginning of the test were measured from two additional replicates per dosage and control. At the end pooled replicates of each dosage were measured. The room temperature was measured continuously by a hygro-thermographe. Light intensities were measured prior to test start.
- Equipement: pH-Meter, pH 191 (WTW)
Hygro-thermographe (KumATHERm)
Quantum Sensor (SKYE INSTRUMENTS LTD)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no, except pH-control
- Photoperiod: 24 h/d light
- Light intensity and quality: mean light intensity was 63.5 µE/m^2*s (minimal 60.0, maximal 68.4 µE/m^2*s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: Cell density was measured via Chlorophyll-a-fluorescence (excitation at 435 nm, emission at 685 nm). Each replicate was measured 6-fold. The cell density was measured at the beginning of the test and every 24 h. Filtrated culture medium was used as ground signal.
- Optical inspection: Microscopic evaluation of the cells was determined at the start and end of the incubation. Alga cells were checked for any unusual cell shapes, color differences, differences in chloroplast morphology, flocculations, adherence of algae to test containers or aggregation of alga cells.
- Equipment: Chlorophyll-innpulsfluorometer (KLEINFELD)
Fluorescent tubes, OSRAM L 36 W /11-860, daylight (test)
Fluorescent tubes, LAMPI 18 W, 60 cm, warmwhite (culture)
Inverse microscope (ZEISS IM 25)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0.1, 1, 10, 100 mg a.i./L
- Results used to determine the conditions for the definitive study: yes

TEST PERFORMANCE
Based on the results of a preliminary range finding test and a first definitive test, the second definitive test was performed with 6 concentration levels in a geometrical series with a factor 2 and 3 replicates each. A control with test medium (without test item, six replicates) was tested under the same conditions as the test group. A pH-control (1000 mg a.i./L) with neutralized stock solution (three replicates) was tested under the same conditions as the test groups. A three day-old preculture incubated at study conditions was used for the main study. Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 h.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

biomass

Remarks on result:

other: adjusted to pH 8.27

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

biomass

Remarks on result:

other: adjusted to pH 8.27

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

253 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: CI 95%: 245 - 260

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

232 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: CI 95%: 226 - 239

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

A summary table on results is given in 'any other information on results'.

Algal growth and biomass production
Inhibition of growth of the freshwater green alga Desmodesmus subspicatus was observed at nominal 250 mg a.i./L (biomass growth and specific growth rate), respectively. The derived EC50-values with 95% confidence intervals for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72h were 232 (226 - 239) and 253 (245 - 260) mg a.i./L, respectively. The NOEC for both biomass growth and specific growth rate was 125 mg a.i./L. Inhibition effects were found to be reversible up to 500 mg a.i./L (all effect levels given as nominal concentrations). With regard to the determinations in the pH-control it can be assumed that effects on the cell growth were caused by pH. This is supported by the pH-control at 1000 mg a.i., adjusted to pH 8.27 where no inhibitory effects were observed. Therefore, the 72h-EC50, adjusted to pH 8.27 > 1000 mg a.i./L. See Overall remark for justification on using pH adjusted effect concentrations as valid effect concentration for the submission substance.

Recovery of Alga Cells
After 72 h algae from the nominal dosage levels 250 - 1000 mg a.i./L and control were transferred to fresh untreated dilution water and allowed to grow for further 4 - 12 d under test conditions. The test item effect was observed to be reversible at 250 - 500 mg a.i./L and not reversible at the concentration 1000 mg a.i./L within 12 days.

Optical evaluation
Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities. At 500- 1000 mg a.i./L only a few alga were found in 1 mL of the replicates after 72 h.

Water quality parameters
Water quality parameters of pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements:
- The temperature during the test was in the range of 21-25 °C.
- The difference from the mean was within ± 2 °C: min.: 22.5 °C, max.: 24.0 °C mean value: 23.3 °C.
- The pH-value in the control replicates increased not higher than 1.0 units: 8.12 - 8.51.)

VALIDITY CRITERIA
The study meets the validity criteria of the guideline (adopted 1984):
- The cell growth increased 49-fold after 72 h in the control (required: 16-fold).

Additional validity criteria of the guideline adopted 2011 (evaluated later):
- The mean coefficient of variation for section-by-section specific growth rates of the control replicates must not exceed 35%: in this study 19%, therefore the study fulfilled the new validity criteria.
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus: in this study 13%, therefore the study did not fulfil the new validity criteria.

Results with reference substance (positive control):

The EC50 - values of the reference item potassium dichromate after 72 h met the validity criteria according to EEC Directive 92/69/EEC Method C.3 Annex 2:
EbC50 (72 h) = 0.49 mg/L
ErC50 (72 h) = 0.94 mg/L

Range of validity
0.20 mg/L < EbC50 (72 h) < 0.75 mg/L
0.60 mg/L < ErC50 (72 h) < 1.03 mg/L

Reported statistics and error estimates:

The NOEC / LOEC were determined by calculation of statistical significance of biomass integrals and growth rates. One Way Analysis of Variance (ANOVA) and DUNNETT'S test (biomass integrals) and the BONFERRONI t-test (growth rates) were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. When running a BONFERRONI t-test an Normality and Homogeneity Test were done first. The a-value for ANOVA and BONFERRONI t-test was a=0.05.

EC50 values and confidence intervals after 72 h were calculated by probit analysis. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TUMPLING 1982).

The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software- SigmaPlot rel. 2000 (2000), SPSS INC.
- SigmaStat rel. 2.03 (1992-1997), SPSS INC.
- Excel rel. 2000 (2000), MICROSOFT CORPORATION
- TOXSTAT rel. 3.4 (1994), Western Ecosystems Technology, Inc., Cheyenne, WY 82001 & David D. Gulley, University of Wyoming, WY, USA

Summary Evaluation after 72 h

Statistically significant differences of biomass integrals and growth rates compared to control values are marked (+), not significant differences are marked (-)

Test item [mg test item/L]	Test item [mg a.i./L]	Replicate	Biomass Integral *	Inhibition of biomass [%]	Growth rate *	Rate-related inhibition [%]
2381	1000	1	-4406	100.00	-0.09	100.00
		2	-4607	100.00	-0.06	100.00
		3	-4613	100.00	-0.11	100.00
		mv	(+)  - 4523	100.00	(+) - 0.08	100.00
1190	500	1	-3742	100.00	-0.09	100.00
		2	-4699	100.00	-0.12	100.00
		3	-4823	100.00	-0.08	100.00
		mv	(+)  - 4422	100.00	(+)  - 0.10	100.00
595	250	1	24380	93.58	0.56	56.43
		2	22915	93.96	0.58	55.48
		3	88559	76.67	0.94	27.58
		mv	(+)  45284	88.07	(+) 0.74	42.72
298	125	1	417334	-9.95	1.37	-5.91
		2	370790	2.31	1.34	-3.34
		3	349428	7.94	1.29	0.69
		mv	(-) 379185	0.10	(-) 1.33	-3.01
149	62.5	1	361676	4.71	1.29	0.46
		2	395327	-4.15	1.32	-1.99
		3	346481	8.72	1.26	2.98
		mv	(-) 367829	3.09	(-)  1.29	0.40
75	31.3	1	405445	-6.82	1.35	-3.96
		2	399222	-5.18	1.33	-2.96
		3	413014	-8.81	1.33	-2.63
		mv	(-) 405895	-6.94	(-)  1.34	-3.18
2381 (adjusted to pH 8.27)	1000 (adjusted to pH 8.27)	1	395783	-4.27	1.32	-2.14
		2	438024	-15.40	1.39	-7.56
		3	466191	-22.82	1.41	-8.68
		mv	(+) § 433334	-14.16	(-)  1.38	-6.23
Control		1	359856		1.27
		2	364040		1.27
		3	378011		1.30
		4	389126		1.30
		5	402801		1.30
		6	383591		1.32
		mv	379571		1.30

mv = mean value; negative inhibitions = increase of growth; § significance caused by increase of growth

* One Way Analysis of Variance (ANOVA), DUNNETT's test (biomass integrals) and the BONFERRONI t-test (growth rate), respectively were carried out for the determination of statistically significant differences compared to control replicates.

Water Parameters

Test item [mg test item/L]	Test item [mg a.i./L]		pH-value
			Start; 0 h	End; 72 h
2381	1000		10.82	8.87
1190	500		10.39	8.47
595	250		9.74	8.23
298	125		9.04	8.19
149	62.5		8.14	8.48
75	31.3		8.15	8.50
2381 (adjusted to pH 8.27)	1000 (adjusted to pH 8.27)		8.27	8.41
Control			8.12	8.51
Temperature: min.: 22.5 °C max.: 24.0 °C mean value: 23.3 °C

DOC Analysis

Test item		Theoretical DOC	DOC [mg/L]
			start, 0h				end, 72h
[mg test item/L]	[mg a.i./L]	[mg/L]	Parallel determination		Mean	RR [%]	Parallel determination		Mean	RR [%]
2381	1000	766	753	754	754	98	750	750	750	98
1190	500	383	376	375	376	98	372	373	373	97
595	250	191	187	187	187	98	187	187	187	98
298	125	95.9	92.5	93	92.8	96	92.3	92.9	92.6	96
149	62.5	47.9	47.7	48	47.9	99	47.9	48	47.9	100
75	31.3	24.1	23	23.6	23.3	95	22.8	23.3	23	95
2381 (adjusted to pH 8.27)	1000 (adjusted to pH 8.27)	766	729	731	730	95	721	722	722	94
Control		-	0.4	0.3	0.3	-	0.1	0.1	0.1	-

RR = Recovery rate (calculated from mean value minus control)

Validity criteria fulfilled:

yes

Remarks:

(adopted 1984, partly for updated guideline from 2011)

Conclusions:

No inhibitory effects on growth was observed at 1000 mg a.i./L adjusted to pH 8.27; therefore, 72h-EC50 (Desmodesmus subspicatus, biomass growth / specific growth rate) > 1000 mg a.i./L, 72h-NOEC (Desmodesmus subspicatus, biomass growth / specific growth rate) >= 1000 mg a.i./L

Executive summary:

The toxicity of the submission substance to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to OECD 201 (adopted 1984) and in compliance with GLP.

The study was conducted under static conditions with an initial cell density of nominally 10^4 cells/mL. Six concentration levels were tested (nominal 31.3 - 62.5 - 125 - 250 - 500 - 1000 mg a.i./L). A control and pH-control (1000 mg a.i./L adjusted to pH 8.27) were run in parallel. Three replicates were tested for each concentration including the pH-control and six replicates for the control. The test item was clearly dissolved in all test replicates throughout the test period.

The concentrations of DOC (dissolved organic carbon) were analysed at all test concentrations at test start and test end. The test concentrations and control were analytically verified via analysis of dissolved organic carbon because HPLC was not applicable to detect the test item concentration applied in this study. Effect concentrations were given based on nominal concentrations, because the recovery rate was >90% for all concentrations.

After 72 hours of exposure, inhibition of growth of the freshwater green alga Desmodesmus subspicatus was observed at nominal 250 mg a.i./L (biomass growth and specific growth rate), respectively. The derived EC50-values with 95% confidence intervals for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72h were 232 (226 - 239) and 253 (245 - 260) mg a.i./L, respectively. The NOEC for both biomass growth and specific growth rate was 125 mg a.i./L. Inhibition effects were found to be reversible up to 500 mg a.i./L within 12 days. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities.

With regard to the determinations in the pH-control it can be assumed that the effects on the cell growth are caused by pH. This is supported by the pH-control at 1000 mg a.i., adjusted to pH 8.27 where no inhibitory effects were observed. According to OECD 23 and ECHA guidance R.7b, pH should be adjusted to lie within the specified range for the test using a suitable buffer, where the substance itself causes a change to the pH of the test medium. This is the case for the test substance. Therefore, the valid 72h-EC50 for algal growth was considered > 1000 mg a.i./L, and 72h-NOEC = 1000 mg a.i./L.

The validity criteria of OECD 201 adopted 1984 were fulfilled. With regard to the updated guideline in 2011, the study fulfilled the new validity criterion for the control of the section-by-section specific growth rate but could not fulfil the criterion of the average specific growth rates during the whole test period in replicate control cultures (re-evaluated with available raw data). However, this violation was not taken as reason to assume significant distortion of results. The study was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.

Description of key information

No inhibitory effects on growth was observed at 1000 mg a.i./L adjusted to pH 8.27; therefore, 72h-EC50 (Desmodesmus subspicatus, biomass growth / specific growth rate) > 1000 mg a.i./L, 72h-NOEC (Desmodesmus subspicatus, biomass growth / specific growth rate) >= 1000 mg a.i./L (nominal, OECD 201, 2002)

Additional information

A reliable study was available on the toxicity of the submission substance to the unicellular freshwater green alga Desmodesmus subspicatus (RL2).The study was performed according to OECD 201 (adopted 1984) and in compliance with GLP.

After 72 hours of exposure, inhibition of growth of the freshwater green alga Desmodesmus subspicatus was observed at nominal 250 mg a.i./L (biomass growth and specific growth rate), respectively. The derived EC50-values with 95% confidence intervals for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72h were 232 (226 - 239) and 253 (245 - 260) mg a.i./L, respectively. The NOEC for both biomass growth and specific growth rate was 125 mg a.i./L. Inhibition effects were found to be reversible up to 500 mg a.i./L within 12 days. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities.

With regard to the determinations in the pH-control it can be assumed that the effects on the cell growth are caused by the high pH. This is supported by the pH-control at 1000 mg a.i., adjusted to pH 8.27 where no inhibitory effects were observed. According to OECD 23 and ECHA guidance R.7b, pH should be adjusted to lie within the specified range for the test using a suitable buffer, where the substance itself causes a change to the pH of the test medium. This is the case for the test substance. Therefore, the valid 72h-EC50 for algal growth was considered > 1000 mg a.i./L, and 72h-NOEC = 1000 mg a.i./L.

The validity criteria of OECD 201 adopted 1984 were fulfilled. With regard to the updated guideline in 2011, the study fulfilled the new validity criterion for the control of the section-by-section specific growth rate but could not fulfil the criterion of the average specific growth rates during the whole test period in replicate control cultures (re-evaluated with available raw data). However, this violation was not taken as reason to assume significant distortion of results. The study was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.