Manganese sulphide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-15 to 2010-03-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted to GLP and to current guidelines. Deviations exist (see overall remarks) but were not considered to have affected the outcome or validity of the test.

Qualifier:

according to guideline

Guideline:

other: US EPA Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms (Method 1002.0)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

yes

Remarks:

different species of invertebrates and shorter time of test

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

different species of invertebrates and shorter time of test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each surviving test group for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 2, 5 and 7 and of the expired test preparations on Days 2, 5, 7 and 8. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

The test item was prepared by stirring an excess (100 mg/L) of the test item in reconstituted water (Elendt M4) using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 25°C for 48 hours. After stirring, any undissolved test item was removed by filtration through a pre-conditioned 0.2 µm Gelman AcroCap filter to produce the 100% v/v saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. Aliquots (50 and 160 mL) of the 100% v/v saturated solution were each separately diluted in a final volume of 500 mL of reconstituted water (Elendt M4) to give the remainder of the test series of 10 and 32% v/v saturated solution. The test solutions were renewed 3 times on days 2, 5 and 7.
Throughout the test the freshly prepared test preparations were observed to be clear, colourless solutions whilst the old media were observed to be pale green dispersions due to the presence of algal cells used as feed for the daphnids.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Common name: Ceriodaphnia dubia
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): 24-27 hours
- Feeding during test: yes
- Food type: suspension of algae (Pseudokirchneriella subcapitata) and YAT (yeast, alfalfa, trout chow) combination
- Amount: each daphnid received 1 to 5 µL of algal suspension and 100 µL of a YAT combination daily
- Frequency: daily

ACCLIMATION
- Acclimation conditions: the same as test conditions
- Type and amount of food: suspension of algae (Pseudokirchneriella subcapitata) and YAT (yeast, alfalfa, trout chow) combination
- Feeding frequency: daily


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The young were at least the third brood of the adults. On the day of test initiation the young were isolated from the single housed cultures within 24 hours of the previous isolation the day before. However, due to the length of preparation of the test media the young were not used within 24 hours as specified by the study plan. Although some of the young may have been up to approximately 27 hours old, it was reasonable to assume that the majority were less than 24 hours old. This deviation was therefore considered not to have affected the outcome or validity of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Hardness:

152 mg/L as CaCO₃ in the control; the water hardness could not be determined for the highest (5.9 mg/L) test concentration due to the metal component of the test item interfering with the test method.

Test temperature:

25°C

pH:

8.0 - 8.2

Dissolved oxygen:

≥ 7.0 mg O₂/L

Salinity:

not reported

Nominal and measured concentrations:

The number of test concentrations was reduced from five, as recommended in the Guideline, to three. The following nominal concentrations were assigned to the definitive test: 10, 32 and 100% v/v saturated solution.
No decline in measured concentration was observed over each media renewal period, the results are based on the mean measured test concentrations as test item only. These were calculated to be 0.54, 2.1 and 5.9 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass vessel
- Type : open (covered to reduce evaporation)
- Material, size, headspace, fill volume: 50 mL glass vessel filled with 15 mL of the test preparation
- Aeration: no
- Renewal rate of test solution: Yes, the test preparations were renewed 3 times on Days 2, 5, and 7.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water (Elendt M4) used for the definitive test was the same as that used to maintain the stock animals.
- Alkalinity: 44 and 40 mg CaCO₃/L in the control and the highest (5.9 mg/L) test group respectively at the start of the test
- Conductivity: 1334 µs/cm in the control and 800 µs/cm in the highest (5.9 mg/L) test group

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 368 to 384 lux

EFFECT PARAMETERS MEASURED: On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Ceriodaphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Ceriodaphnia as compared to the controls. The number of Ceriodaphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Ceriodaphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: The conditions for the definitive study are based an acute toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 2702/0146).

Reference substance (positive control):

no

Duration:

8 d

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% CL=0.97 - 1.4 mg/l

Duration:

8 d

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL=0.95 - 2.1 mg/l

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Details on results:

- Mortality of parent animals: Mortality (immobilisation) occurred predominantly at the highest test concentrations of 2.1 and 5.9 mg/L resulting in 100% and 80% mortality by Day 8 respectively. Slightly greater toxicity was observed at the test concentration of 2.1 mg/L compared to the higher test concentration of 5.9 mg/L. A review of the data did not reveal a reason for this given that no unexpected analytical results were obtained indicating that no errors in media preparation occurred. It was therefore considered that this effect was possibly due to natural variation within the daphnid population.

- No. of offspring produced per day per female: After 8 days there were no statistically significant differences between the control and 0.54 mg/L test group (25 and 27 live young per female respectively). The 5.9 mg/L test group showed a statistically significant difference from the control and the 0.54 mg/L test group after 8 days in terms of producing fewer numbers of live young per adult (15 young/female). The 2.1 mg/L test group data was not included in statistical analysis as exposure to the test item eliminated all the daphnids prior to Day 8 of the test.

- Morphological abnormalities: There was a significant effect on size and colour of the daphnids at the test concentration of 5.9 mg/L in that some of the daphnids were markedly smaller and paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.

- Time to first brood release or time to hatch: Young were first produced in the control test group on Day 3 of the test. Numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation. Young daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

- Egg development time: 3-4 days

The observations for each test and control group are summarised in Tables 1 to 5. The total cumulative production of live young is given in Table 6 and the number of live young produced per adult is shown in Table 7.

Table 1. Summary of Findings Following the Exposure of Ceriodaphnia dubia for 8 Days

Mean Measured		Number of		Number of		Number of
Concentration	%	Live Young*		Dead Young		Unhatched Eggs
(mg/L as Test	Survival		Per Female		Per Female		Per Female
Item)	of P1	Total	(cumulative)	Total	(cumulative)	Total	(cumulative)
Control	90	227	25	0	0	0	0
0.54	100	273	27	0	0	0	0
2.1	0	0	0	0	0	0	0
5.9	20	29	15	2	<1	0	0

Table 2. Summary of Observations of the Control Group

	Adults	Number of Adults
Day	Surviving	with Eggs in Brood	Live Young	Dead Young	Unhatched Eggs
		Pouch
1	10	0	0	0	0
2	10	8	0	0	0
3	9	8	4	0	0
4	9	9	25	0	0
5	9	9	68	0	0
6	9	9	64	0	0
7	9	9	0	0	0
8	9	9	66	0	0
		TOTALS	227	0	0

Table 3. Summary of Observations of the 0.54 mg/L Test Group 

	Adults	Number of Adults
Day	Surviving	with Eggs in Brood	Live Young	Dead Young	Unhatched Eggs
		Pouch
1	10	0	0	0	0
2	10	6	0	0	0
3	10	10	4	0	0
4	10	10	31	0	0
5	10	10	73	0	0
6	10	10	35	0	0
7	10	10	39	0	0
8	10	10	91	0	0
		TOTALS	273	0	0

Table 4. Summary of Observations of the 2.1 mg/L Test Group

	Adults	Number of Adults
Day	Surviving	with Eggs in Brood	Live Young	Dead Young	Unhatched Eggs
		Pouch
1	10	0	0	0	0
2	9	2	0	0	0
3	6	5	0	0	0
4	2	2	5	0	0
5	0	0	0	0	0
		TOTALS	5	0	0

Table 5. Summary of Observations of the 5.9 mg/L Test Group

	Adults	Number of Adults
Day	Surviving	with Eggs in Brood	Live Young	Dead Young	Unhatched Eggs
		Pouch
1	10	0	0	0	0
2	7	3	0	0	0
3	7	5	0	0	0
4	6	5	11	0	0
5	5	5	21	2	0
6	5	5	25	0	0
7	2	2	10	0	0
8	2	2	0	0	0
		TOTALS	67	2	0

Table 6. Total Cumulative Production of Live Young

Mean Measured				Day
Concentration
(mg/L as Test	1	2	3	4	5	6	7	8
Item)
Control	0	0	4	29	97	161	161	227
0.54	0	0	4	35	108	143	182	273
2.1	0	0	0	5	5	A/D
5.9	0	0	0	11	32	57	67	67

Table 7. Number of Live Young Produced per Adult (Non-Cumulative)

Mean Measured				Day
Concentration
(mg/L as Test	1	2	3	4	5	6	7	8
Item)
Control	0	0	0	3	8	7	0	7
0.54	0	0	0	3	7	4	4	9
2.1	0	0	0	1	0	A/D
5.9	0	0	0	2	4	3	1	1

Physico-Chemical Measurements (Recorded in One Replicate Test Vessel) of Oxygen (mg O₂/L), Temperature (°C) and pH

Mean Measured Concentration (mg/L as Test Item)		Day
		0	1	2*	2+	3	4	5*	5+
Control	O2	9.0	-	7.6	7.8	-	-	7.0	7.6
	%^D	107	-	92	94	-	-	84	92
	T°C**	24	25	25	25	26	25	25	25
	pH	8.3	-	8.2	8.2	-	-	8.2	8.2
0.54	O2	8.8	-	7.6	8.0	-	-	7.1	7.6
	%^D	105	-	92	96	-	-	86	92
	T°C**	24	25	25	25	26	25	25	25
	pH	8.2	-	8.1	8.1	-	-	8.2	8.2
2.1	O2	9.1	-	7.4	8.0	-	-	7.1	7.8
	%^D	108	-	89	96	-	-	86	94
	T°C**	24	25	25	25	26	25	25	25
	pH	8.2	-	8.0	8.1	-	-	8.2	8.1
5.9	O2	8.9	-	7.6	7.8	-	-	7.3	7.1
	%^D	106	-	92	94	-	-	88	86
	T°C**	24	25	25	25	26	25	25	25
	pH	8.0	-	8.0	8.1	-	-	8.2	8.0

Mean Measured Concentration (mg/L as Test Item)		Day
		6	7*	7+	8
Control	O2	-	8.0	8.1	8.0
	%^D	-	96	98	96
	T°C**	25	25	25	25
	pH	-	8.1	8.1	8.2
0.54	O2	-	8.1	8.1	8.0
	%^D	-	98	98	96
	T°C	25	25	25	25
	pH	-	8.1	8.1	8.2
2.1	O2
	%^D		A/D
	T°C
	pH
5.9	O2	-	8.0	8.0	8.0
	%^D	-	96	96	96
	T°C	25	25	25	25
	pH	-	8.0	8.0	8.2

*Before renewal

+After renewal

^DASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

**Mean of temperature readings taken from Control R₁and 5.9 mg/L R₁

*Before renewal

+After renewal

^DASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

**Mean of temperature readings taken from replicates Control R₁and 2.1 mg/L R₁(second reading on Day 5 taken from 5.9 mg/L Replicate R₁due to 100% mortality at 2.1 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The No Observed Exposure Concentration (NOEC) is 0.54 mg/L .

Executive summary:

The long-term toxicity of the test material to aquatic invertebrates was assessed using a method similar to OECD Test Guideline 211 and in compliance with GLP.

Mortality (immobilisation) of parents occurred predominantly at the highest test concentrations of 2.1 and 5.9 mg/L resulting in 100 % and 80 % mortality by Day 8 respectively. Slightly greater toxicity was observed at the test concentration of 2.1 mg/L compared to the higher test concentration of 5.9 mg/L. A review of the data did not reveal a reason for this given that no unexpected analytical results were obtained indicating that no errors in media preparation occurred. It was therefore considered that this effect was possibly due to natural variation within the daphnid population.

After 8 days there were no statistically significant differences between the control and 0.54 mg/L test group in the number of offspring produced per day per female (25 and 27 live young per female respectively). The 5.9 mg/L test group showed a statistically significant difference from the control and the 0.54 mg/L test group after 8 days in terms of producing fewer numbers of live young per adult (15 young/female). The 2.1 mg/L test group data was not included in statistical analysis as exposure to the test item eliminated all the daphnids prior to Day 8 of the test.

There was a significant effect on size and colour of the daphnids at the test concentration of 5.9 mg/L in that some of the daphnids were markedly smaller and paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.

Young were first produced in the control test group on Day 3 of the test. Numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation. Young daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Under the conditions of the study the No Observed Exposure Concentration (NOEC) is 0.54 mg/L.  

Description of key information

The No Observed Exposure Concentration (NOEC) is 0.54 mg/L .

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.54 mg/L

Additional information

The long-term toxicity of the test material to aquatic invertebrates was assessed using a method similar to OECD Test Guideline 211 and in compliance with GLP.

Mortality (immobilisation) of parents occurred predominantly at the highest test concentrations of 2.1 and 5.9 mg/L resulting in 100 % and 80 % mortality by Day 8 respectively. Slightly greater toxicity was observed at the test concentration of 2.1 mg/L compared to the higher test concentration of 5.9 mg/L. A review of the data did not reveal a reason for this given that no unexpected analytical results were obtained indicating that no errors in media preparation occurred. It was therefore considered that this effect was possibly due to natural variation within the daphnid population.

After 8 days there were no statistically significant differences between the control and 0.54 mg/L test group in the number of offspring produced per day per female (25 and 27 live young per female respectively). The 5.9 mg/L test group showed a statistically significant difference from the control and the 0.54 mg/L test group after 8 days in terms of producing fewer numbers of live young per adult (15 young/female). The 2.1 mg/L test group data was not included in statistical analysis as exposure to the test item eliminated all the daphnids prior to Day 8 of the test.

There was a significant effect on size and colour of the daphnids at the test concentration of 5.9 mg/L in that some of the daphnids were markedly smaller and paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.

Young were first produced in the control test group on Day 3 of the test. Numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation. Young daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Under the conditions of the study the No Observed Exposure Concentration (NOEC) is 0.54 mg/L.

With respect to classification and labelling the chronic ERV value has been derived from the chronic fish study conducted on the soluble manganese salt manganese sulphate. The chronic ERV has been compared to the transformation dissolution data. This approach is in accoradance with CLP guidance.