Hydroxylammonium chloride

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:

3 - 6 April 2017

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:

adopted 2006, corrected 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: samples of test media were analysed at 0 (new media), 24, 48 and 72 h. At the start of the test, 10 mL samples of freshly prepared test media were taken from the control and each test media preparation flask for chemical analysis. At 24 and 48 h, samples were taken from each replicate test vessel from the control and each test concentration and pooled before analysis. At 72 h, inoculated replicate test vessels at each treatment level were pooled and sampled. Although samples were taken for analysis after 48 h exposure, the analytical results have not been reported since the analytical recovery samples failed the acceptance criteria and the results were therefore considered unreliable. Re-analysis of the batch was not considered possible due to the instability of the test substance in the media.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 50 mg of test substance was dissolved in 500 mL of EC medium to give a 100 mg/L stock solution. Dilutions were prepared in EC medium, to give the final test concentrations. A control treatment was prepared only with EC medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., UK (concentrated liquid slope culture was stored at 2 -8 °C. A typical shelf life for each slope was 8 months, after which the slopes were discarded)
- Age of inoculum (at test initiation): at least 72 h
- Method of cultivation: a sub-sample (ca 200 µL) of a current liquid slope culture was added to two flasks containing 100 mL EC medium and incubated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

-

Test temperature:

23.7 - 24.5 ºC

pH:

Control: 7.61 - 10.01
Test concentrations: 7.12 - 9.73

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal: control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Geom. mean meas.: < LOQ, 0.050, 0.063, 0.13, 0.35 and 3.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile autoclaved 250 mL conical flasks
- Type: all flasks were loosely-capped
- Material, size, headspace, fill volume: fill volume: 100 mL, material: glass
- Initial cells density: 1 x 10^4 algae cells/mL
- Control end cells density: 192 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (EC Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the dilution media used was EC medium and was prepared according to the guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: the pH and appearance of the test substance in the test media were recorded at the start and end of the test. The temperature was recorded continuously using a digital temperature logger.

OTHER TEST CONDITIONS
- Photoperiod: constant light
- Light intensity and quality: 4440-8880 Lux, florescent lights (400 - 700 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: at 24 h intervals pre-determined volumes of test media were removed from each test vessel and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: control, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: based on the results of the range-finding test, the definitive test was conducted at nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.20 – 0.23 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.075 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.062 – 0.083 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes (the control cell densities increased by a factor of 191)
- Colour differences: the freshly prepared test media at 0 h appeared as a colourless solution. The colour of the test media following the 72 h exposure period was green in the control and 0.05 mg/L concentration, light green in the 0.063 mg/L concentration, pale green in the 0.13 mg/L concentration and colourless in the 0.35 and 3.9 mg/L concentrations.

VALIDITY CRITERIA

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The control cell densities increased by a factor of 191	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rates (0-24, 24-48 and 48-72 h) in the control culture was 6.56%	yes
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. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation for average specific growth rates during the whole test period in replicate control culture was 2.19%	yes

BIOLOGICAL RESULTS

Table 2: Growth of Pseudokirchneriella subcapitata cultures during the definitive test.

Geometric mean measured concentration (mg/L)	Mean cell concentration (cells x 10^4/mL)
	0-24 h	0-48 h	0-72 h
Control	8.32	47.5	192
0.05	6.66	42.7	197
0.063	4.46	27.1	142
0.13	1.41	6.84	42.7
0.35	1.28	1.77	2.64
3.9	1.11	1.42	1.43

Table 3: Inhibition (%) in growth of Pseudokirchneriella subcapitata relative to the control treatment.

Geometric mean measured concentration (mg/L)	Reduction in average specific growth rates relative to the control (%)
	0-24 h	0-48 h	0-72 h
Control	-	-	-
0.05	10	3	0
0.063	30	14	6
0.13	84	50	29
0.35	89	85	82
3.9	95	91	93

Table 4: Further toxicity data

Parameter	Toxicity values (mg/L) with 95% confidence limits
	72-hour
Yield
EyC10	0.055 (NC – 0.057)
EyC20	0.06 (0.016 – 0.065)
EyC50	0.092 (0.075 – 0.10)
NOEC	0.05
Biomass
EbC10	0.053 (NC – 0.056)
EbC20	0.057 (0.029 – 0.061)
EbC50	0.086 (0.074 – 0.095)
NOEC	0.05
Growth Rate
ErC20	0.10 (0.092 – 0.11)
NOEC	0.05

ANALYTICAL RESULTS

Table 5: Analytical results of the test media.

Nominal concentration (mg/L)	Measured concentration (mg/L)
	0 h (New media)	% nominal	24 h (Old media)	% nominal	72 h (Old media)	% nominal	Geometric mean measured concentration (mg/L)
Control	<LOQ	-	<LOQ	-	<LOQ	-	-
0.1	<LOQ	-	<LOQ	-	<LOQ	-	0.05
0.32	0.0995	31	<LOQ	-	<LOQ	-	0.063
1	0.856	86	<LOQ	-	<LOQ	-	0.13
3.2	2.53	79	0.346	11	<LOQ	-	0.35
10	9.78	98	4.13	41	1.5	15	3.9

Where measured concentrations were <LOQ, a value of half the LOQ (0.050 mg/L) was used in the calculation of the geometric mean measured concentration given that peaks were observed in the samples but could not be quantified.

Although samples were taken for analysis after 48 h exposure, the analytical results have not been reported since the analytical recovery samples failed the acceptance criteria and the results were therefore considered unreliable. Re-analysis of the batch was not considered possible due to the instability of the test substance in the media.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErC50 (72 h) = 0.21 mg/L (geom. mean meas., P. subcapitata, OECD 201)

ErC10 (72 h) = 0.075 mg/L (geom. mean meas., P. subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.21 mg/L

EC10 or NOEC for freshwater algae:

0.075 mg/L

Additional information

A study is available, in which the toxicity of the substance hydroxylammonium chloride (CAS 5470-11-1) to aquatic algae was assessed. The study was conducted according to OECD guideline 201 and GLP. Pseudokirchneriella subcapitata was used as test organism. The green algae was exposed to nominal test item concentrations of 0.10, 0.32, 1.0, 3.2, 10 mg/L and a control (corresponding to geom. mean meas. concentrations of 0.050, 0.063, 0.13, 0.35 and 3.9 mg/L) for 72 h. An analytical monitoring via LC-TOF/MS was performed at 0, 24 and 72 h in order to verify the concentrations in all test item concentrations and in the control. All validity criteria given by the guideline were fulfilled. The study resulted in an ErC50 (72 h) of 0.21 mg/L and an ErC10 (72 h) of 0.075 mg/L (both geom. mean meas).