The toxic effects of three dinoflagellate species from the genus Karenia on invertebrate larvae and finfish

Figures & data

Table 1  Toxicity of Karenia species to juvenile Chinook salmon (Oncorhynchus tshawytscha) and juvenile New Zealand snapper (Pagrus auratus).

Fish tested	Karenia species	Cell density (10^6 cells L^–1)	LT50 ^1 (hours)
Salmon	K. brevis	0.18	0.75
	K. brevis	0.9	0.42
	K. brevis	4.6	0.17
	K. brevisulcata	1.3	0.93
	K. brevisulcata	11	0.48
Snapper	K. mikimotoi	3.4^2	N^3
	K. brevisulcata	16.1^2	7.5
	K. brevis	5.9^2	4.5
Notes: n = 4 for all experiments; ^1least time to 50% fish being moribund; ^2final cell density after step-wise addition of 8 L of microalgal mass culture over 2.5 h; ^3no morbidity after 24 h observation.

Table 2  Toxic effects of fresh cultures (19–28 days) and solid phase extraction (SPE)¹ extracts of Karenia species and of brevisulcatic acid (BSX)² toxins, on invertebrate larvae³ (6 days old) over 1–24 h observation.

Karenia species	Concentration	Invertebrates tested	Affected
K. brevis	0.1 × 10^6 cells L^–1	All	None
	0.9 × 10^6 cells L^–1	All	None
	8.7 × 10^6 cells L^–1	All	None
	SPE extract 9 × 10^6 cells L^–1	GM	No
K. brevisulcata	0.8 × 10^6 cells L^–1	All	None
	8.4 × 10^6 cells L^–1	All	GM, sea urchin, paua
	84 × 10^6 cells L^–1	All	All except brine shrim
	Cell–free (56 d)	GM	Yes
	SPE extract 72 × 10^6 cells L^–1	GM	Yes
	BSX toxins 1–100 μg L^–1	GM	No
K. mikimotoi	0.5 × 10^6 cells L^–1	All except paua	None
	6 × 10^6 cells L^–1	All except paua	None
	56 × 10^6 cells L–1	All except paua	None
	1.2 × 10^6 cells L^–1	Paua	No
	12 × 10^6 cells L^–1	Paua	Yes
	118 × 10^6 cells L^–1	Paua	Yes
	SPE extract 85 × 10^6 cells L^–1	GM	No
Notes: There were 3 replicates of 6–10 larvae for all experiments; ^1SPE of culture using polymeric absorbent; ^2BSX-1, -2, -4, and -5; ^3brine shrimp, Greenshell^TM mussel (GM), Pacific oyster, Paua (New Zealand abalone), Sea slug and Sea urchin.

Figure 1  Phycotoxins produced by Karenia brevis and Karenia brevisulcata—batch carboy cultures grown for 19–20 days and extracted by solid phase extraction for liquid chromatography-mass spectrometry determination; brevisulcatic acid (BSX) concentrations in brevetoxin-2 (BTX-2) response equivalents. A, Karenia brevis. B, Karenia brevisulcata. Columns and error bars depict the mean ± SEM (n = 3); note different scales on y-axes.

Figure 2  Responses of juvenile salmon (Oncorhynchus tshawytscha) to two Karenia species versus time: K. brevis 4.6 × 10⁶ cells L^–1 (▴) and K. brevis 0.9×10⁶ cells L^-1 (♦), K. brevisulcata 11×10⁶ cells L^-1 (□) and K. brevisulcata 1.3×10⁶ cells L^-1 (▪).

Notes: The points/bars depict the mean ± SEM (n = 4). Health index: 0, death; 1, extreme loss of balance; 2, leaping; 3, gasping; 4, agitation; 5, slight loss of balance; 6, healthy.

Figure 3  Responses of marine invertebrate larvae (six days old) to Karenia cultures over 24 h. A, Response of paua (Haliotis iris) to K. mikimotoi: control (sterile seawater [×]); 12 × 10⁶ cells L^–1 (□) and 118 x 10⁶ cells L^–1 (♦). B, Greenshell^TM mussel (Perna canaliculus). C, Pacific oyster (Crassostrea gigas). D, Sea slug (Pleurobranchia maculata). E, Sea urchin (Evechinus chloroticus). F, Paua. B–F, Responses of species to K. brevisulcata: control (sterile seawater [×]); 8.4 × 10⁶ cells L^-1 (□); 84 × 10⁶ cells L^-1 (♦).

Notes: Points and error bars depict the mean ± SEM (n = 3); health scores: 0, death; 1, moribund; 2, severe stress; 3, stress; 4, healthy but larvae static; 5, healthy and larvae swimming; median for 6–10 larvae in each of the triplicate wells.

Figure 4  Greenshell^TM mussel (Perna canaliculus) larvae (six days old) affected by Karenia brevisulcata during exposure over 24 h to 8.4 × 10⁶ cells L^–1. A, Healthy and static, score 4. B, Stressed, the velum started to disintegrate, and cilia withdrawn but healthy and moving, score 3. C, Severely stressed, larvae retracted into shell and cilia moving inside shell, score 2. D, Moribund, cilia gone, larvae disintegrating, still some jerking movement, score 1. E, Dead, disintegrated larvae appeared amorphous, decayed and jelly-like, and cells were being discharged from the dead animal, score 0.

Notes: Black arrow in A–C shows cilia and white arrow in E shows leaked tissue.

Table 3  Toxicity of cultures of Karenia species to invertebrate larvae.

				LC50 (×10^6 cells L^–1)
Culture	Invertebrate	LT50 (h)	Concentration (×10^6 cells L^–1)	2 h	24 h
K. brevisulcata	P. oyster^2	9.2 ± 1.5	34	>35	10
	GM^3	1.9 ± 0.5	7.5	10	2
	Sea slug	6^1	84	>90	10
	Sea urchin	6.1 ± 0.3	7.8	12	2
	Paua	1^1	9.6	5	2
	Brine shrimp	>24	84	>90	>90
K. mikimotoi	P. oyster^2	>24	100	>100	>100
	GM^3	>24	39	>40	>40
	Sea slug	>24	22	>25	>25
	Sea urchin	>24	64	>70	>70
	Paua	10.0 ± 2.2	12	20	2
	Brine shrimp	>24	56	>60	>60
K. brevis	P. oyster^2	>24	7	>7	>7
	GM^3	>24	13	>13	>13
	Sea slug	>24	3	>3	>3
	Sea urchin	>24	13	>13	>13
	Paua	>24	13	>13	>13
	Brine shrimp	>24	8.7	>9	>9
Notes: LT50, least time for 50% of larvae to become moribund after exposure to specified cell concentration (mean ± SEM from curve fit); LC50, lowest cell concentration for 50% of larvae to become moribund after exposure time of 2 or 24 h (estimate; 50% moribund defined as mean toxicity score of 2.5); >, survival of all larvae at longest observation time or highest cell concentration used; ^1estimate (insufficient data for curve fit); ^2Pacific oyster; ^3Greenshell^TM mussel.

Figure 5  Responses of Greenshell^TM mussel (Perna canaliculus) larvae to a 56-day K. brevisulcata mass culture without living cells □ and solid phase extraction extracts of 20–30-day Karenia mass cultures: K. mikimotoi (× [85 × 10⁶ cells L^–1]), K. brevis (□ [9 × 10⁶ cells L^–1]) and K. brevisulcata (♦ [72 × 10⁶ cells L^–1]).

Note: Points and error bars depict the mean ± SEM (n = 3).