2-aminopyridin-3-ol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-05 to 2016-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-aminopyridin-3-ol

EC Number:

240-886-8

EC Name:

2-aminopyridin-3-ol

Cas Number:

16867-03-1

Molecular formula:

C5H6N2O

IUPAC Name:

2-aminopyridin-3-ol

Details on test material:

Name of test material (as cited in the report): A132 2-Amino-3-hydroxypyridine

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). Due to 100% mortality after 24 hours in all replicates of the highest concentration level of 100 mg/L, the measured values of the highest concentration level during the second exposure interval (24 and 48 hours) were excluded from evaluation.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test item concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels.

Range (target)
Recoveries of the test item should be within ± 20% of the nominal concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water prior to each exposure interval (0 and 24 hours). The stock solution was treated with ultrasound for 20 minutes at room temperature and stirred thereafter at approximately 1100 rpm for 30 minutes at room temperature.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by diluting the stock solution of 100 mg/L with dilution water, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
This corresponds to geometric mean measured test item concentrations of: 5.44 - 10.2 - 22.2 - 43.7 - 93.5 mg/L
The test concentrations were selected based on the results of a non-GLP preliminary range finding test.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Quality Parameters of the Dilution Water at the Start of the Exposure Intervals (0 and 24 hours)
Dilution water dated: pH-Value Dissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
0 hours: 2016-10-05 7.68 8.20 19.9 492 175
24 hours: 2016-10-06 7.62 8.12 20.6 500 180

Test temperature:

see above

pH:

Water Quality Parameters at the Start of the Exposure Intervals (0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)

Geometric mean
measured concentration
of the test item
[mg/L] 0 hours 24 hours
pH-value Dissolved O2 concentration [mg/L] pH-value Dissolved O2 concentration [mg/L]
93.5 7.69 6.55 7.52 7.07
43.7 7.70 7.42 7.54 7.54
22.2 7.73 7.89 7.57 7.60
10.2 7.74 8.01 7.58 7.86
5.44 7.64 8.01 7.60 7.49
Control 7.68 8.20 7.62 8.12


Water Quality Parameters at the End of the Exposure Interval (24 hours)
(measured in all replicates(containing daphnids) per concentration level and control)

Geometric mean
measured concentration
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
93.5 7.54 7.57 7.56 7.58 6.34 6.42 6.66 6.60
43.7 7.55 7.55 7.55 7.55 6.81 6.53 6.82 6.38
22.2 7.54 7.53 7.55 7.54 7.31 6.46 6.91 6.91
10.2 7.55 7.56 7.55 7.53 6.55 7.09 6.68 6.73
5.44 7.53 7.54 7.54 7.53 7.23 7.22 7.09 6.88
Control 7.56 7.54 7.56 7.53 6.92 7.31 6.60 7.11

 

Water Quality Parameters at the End of the Exposure Interval (48 hours)
(measured in all replicates(containing daphnids) per concentration level and control)

Geometric mean
measured concentration
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
93.5 — — — — — — — —
43.7 — — — 7.62 — — — 6.72
22.2 7.55 7.55 7.59 7.60 7.46 7.40 7.47 7.55
10.2 7.51 7.51 7.54 7.53 7.48 7.44 7.68 7.36
5.44 7.35 7.34 7.37 7.46 7.61 7.74 7.30 7.35
Control 7.21 7.31 7.31 7.31 7.11 7.33 7.26 7.40
— = not determined, due to 100% mortality after 24 hours

Dissolved oxygen:

see above

Conductivity:

see above

Nominal and measured concentrations:

nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
geometric mean measured concentrations: 5.44 - 10.2 - 22.2 - 43.7 - 93.5 mg/L

Details on test conditions:

Test method
The study was performed with a semi-static test design (with a renewal of the test solutions after 24 hours) as a worst-case exposure.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatisation
Acclimatisation of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

20.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 17.6 - 24.2

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

19.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: < 5.44 - 22.1

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

28.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 25.5 - 34.3

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

24.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 22.4 - 38.6

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

93.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

43.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2016 09-13 to 2016-09-14.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.03 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.17 - 3.63 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EC100-values (after 24 and 48 hours) were empirically derived from the observation data. All effect levels (EC10 / 50 / 100) given are based on the geometric mean measured test item concentrations, since the measured test item concentrations were not within ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours is shown graphically.

EC-values and statistical analyses
The EC10- and the EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression with the software GraphPad Prism5. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The concentration-effect relationships after 24 and 48 hours are shown graphically.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

A132 2-Amino-3- hydroxypyridine Geometric mean measured test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
93.5	100	100	100	100	100	100% mortality after 24 hours
43.7	100	100	100	80	95	100% mortality after 24 hours			100	100
22.2	20	40	0	0	15	20	60	0	20	25
10.2	0	0	0	0	0	0	20	0	0	5
5.44	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

A132 2-Amino-3- hydroxypyridine Geometric mean measured test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
93.5	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
43.7	5 / 5	5 / 5	5 / 5	4 / 5	19 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
22.2	1 / 5	2 / 5	0 / 5	0 / 5	3 / 20	1 / 5	3 / 5	0 / 5	1 / 5	5 / 20
10.2	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	1 / 5	0 / 5	0 / 5	1 / 20
5.44	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

 Additional Observations during the Definitive Test

 

All tested concentration levels were visually clear throughout the exposure. During the exposure intervals, the color of the solutions changed from brownish to red-brown in the concentration level 100 mg/L and from colorless to light brownish in the concentration level of 12.5 mg/L. The color was concentration-related. The concentration level 6.25 mg/L was colorless throughout the exposure intervals.

 

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item A132 2-Amino-3-hydroxypyridine were analytically verified via HPLC-DAD at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and in the control.

The measured concentrations of the test item were in the range of 88 to 107% of the nominal values at the start of the exposure intervals (0 and 24 hours) and 69 to 104% at the end of the exposure intervals (24 and 48 hours).

The geometric mean measured concentrations of the test item A132 2‑Amino-3-hydroxypyridine are: 5.44 - 10.2 - 22.2 - 43.7 - 93.5 mg/L.

 

Measured Concentrations and Percent of Nominal Concentration of the Test ItemA132 2‑Amino-3-hydroxypyridine during the Definitive Test

Sampling date	2016-10-05 Start of the  exposure interval 0 hours		2016-10-06 End of the  exposure interval 24 hours		2016-10-06 Start of the  exposure interval 24 hours		2016-10-07 End of the  exposure interval 48 hours
Start of analysis	2016-10-05		2016-10-06		2016-10-06		2016-10-07
Nominal test item concentration   [mg/L]	A132 2-Amino-3-hydroxypyridine								Geometric mean measured test item concentration [mg/L]
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	100	100	87.5	88	(95.4)	(95)	(71.6)	(72)	93.5
50.0	49.6	99	44.3	89	47.8	96	34.7	69	43.7
25.0	23.3^1)	93	26.1	104	23.3	93	17.2	69	22.2
12.5	12.3	99	9.41^2)	75	11.0	88	8.57	69	10.2
6.25	6.68	107	5.34	85	5.54	89	4.45	71	5.44
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc.= measured concentration of the test item, single injection, dilution factors taken into account

%                  = percent of the nominal concentration of the test item

LOQ             = limit of quantification of the analytical method (0.6 mg/L of the test item)

¹⁾                   =reanalysedon16-10-06, mean value of 2 replicates

²⁾                   =reanalysedon16-10-07, mean value of 3 replicates

(…)               = Due to 100% mortality after 24 hours, the measured concentrations during the 2^ndexposure interval of the stock solution are given only for information. These values were not used for calculation of the geometric mean measured concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item A132 2-Amino-3-hydroxypyridine, the 48 hour-EC50 for Daphnia magna was 24.6 mg/L (95% confidence limits: 22.4 - 38.6 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item A132 2‑Amino-3-hydroxypyridine (batch number:20150502) were determined at the test facility according to OECD 202 (2004) from 2016-10-05 to 2016-10-07.

The study was conducted under semi-static conditions over a period of 48 hours with 5 concentrations of the test item A132 2-Amino-3-hydroxypyridine in the range of 6.25 - 100 mg/L, prepared in a geometric series with a separation factor of 2.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test item A132 2-Amino-3-hydroxypyridine were analytically verified via HPLC-DAD at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and in the control.

The measured concentrations of the test item were in the range of 88 to 107% of the nominal values at the start of the exposure intervals (0 and 24 hours) and 69 to 104% at the end of the exposure intervals (24 and 48 hours).

The geometric mean measured concentrations of the test itemA132 2‑Amino-3-hydroxypyridine are: 5.44 - 10.2 - 22.2 - 43.7 - 93.5 mg/L.

Since the measured test item concentrations were not within the range of±20% of the nominal concentrations, the effect levels given are based on the geometric mean measured concentrations of the test item A132 2-Amino-3-hydroxypyridine.

The validity criteria of the test guidelines were fulfilled.

 

 EC₁₀-, EC₅₀-(with 95% Confidence Limits), EC₁₀₀-Values

(based on the geometric mean measured concentrations of the test item)

A132 2-Amino-3-hydroxypyridine
Effect levels	Test duration [h]	Geometric mean measured test item concentrations [mg/L]
EC10 (with 95% confidence limits)	24	20.8	(Cl: 17.6 - 24.2)
	48	19.8	(Cl: < 5.44 - 22.1)
EC50 (with 95% confidence limits)	24	28.5	(Cl: 25.5 - 34.3)
	48	24.6	(Cl: 22.4 - 38.6)
EC100	24	93.5
	48	43.7